Novel T-cell membrane protein (TIRC7), peptides and antibodies derived therefrom and uses thereof

ABSTRACT

Described are generally a T cell immune response cDNA 7 (TIRC7) encoding a novel T-cell transmembrane protein as well as peptides und polypeptides derived therefrom and antibodies recognizing said (poly)peptides. More particularly, peptide and polypeptide as well as antibodies being capable of inhibiting T-cell stimulation through the T-cell membrane protein (TIRC7) are provided. Furthermore, vectors comprising the aforementioned polynucleotides and host cells transformed therewith as well as their use in the production of the above-defined proteins, peptides or polypeptides are described. Additionally, pharmaceutical and diagnostic compositions are provided comprising any one of the afore described polynucleotide, vector, protein, peptide, polypeptide, or antibody. Furthermore, methods and uses for modulating immune responses through the novel TIRC7 membrane protein as well as pharmaceutical compositions comprising agents which act on the TIRC7 membrane protein or its ligand are described. Also, the use of said polynucleotide, vector, protein, peptide, polypeptide, or antibody for the preparation of pharmaceutical compositions for use in organ transplantation, for the treatment of autoimmune, allergic or infectious diseases, or for treatment of tumors is provided.  
     Furthermore, methods for modulating (antigen-specific) T cell unresponsiveness inducing maintaining or reversing T cell unresponsiveness by inhibiting or stimulating an (unresponsive) T cell through the novel TIRC7 membrane protein are encompassed herein.

FIELD OF THE INVENTION

[0001] The present invention pertains generally to a T cell immuneresponse cDNA 7 (TIRC7) encoding a novel T-cell transmembrane protein aswell as peptides und polypeptides derived therefrom and antibodiesrecognizing said (poly)peptides. In a first aspect, the presentinvention relates to TIRC7 cDNA and its encoded protein. In a furtheraspect, the present invention relates to polynucleotides derived fromsaid TIRC7 cDNA encoding a peptide or polypeptide being capable ofinhibiting T-cell stimulation through the T-cell membrane protein(TIRC7). Furthermore, the present invention relates to vectorscomprising such polynucleotides and host cells transformed therewith aswell as their use in the production of the above-defined peptides orpolypeptides. In addition, the present invention relates to the(poly)peptide encoded by said polynucleotides or obtainable by themethod of the invention. In another important aspect the presentinvention relates to antibodies against said peptides and polypeptidesthat are capable of inhibiting T-cell stimulation through the T-cellmembrane protein (TIRC7). The present invention additionally relates topharmaceutical and diagnostic compositions comprising the aformentionedpeptide, polypeptide, or antibody. Furthermore, the present inventionrelates to methods and uses for modulating immune responses through thenovel TIRC7 membrane protein as well as to pharmaceutical compositionscomprising agents which act on the TIRC7 membrane protein or its ligand.Also, the invention relates to the use of the before-describedpolynucleotide, vector, peptide, polypeptide, or antibody for thepreparation of pharmaceutical compositions for use in organtransplantation, for the treatment of autoimmune, allergic or infectiousdiseases, or for treatment of tumors. Furthermore, the present inventionrelates to methods for modulating (antigen-specific) T cellunresponsiveness. The present invention encompasses methods forinducing, maintaining or reversing T cell unresponsiveness by inhibitingor stimulating an (unresponsive) T cell through the novel TIRC7 membraneprotein.

[0002] Several documents are cited throughout the text of thisspecification. Each of the documents cited herein (including anymanufacturer's specifications, instructions, etc.) are herebyincorporated herein by reference; however, there is no admission thatany document cited is indeed prior art as to the present invention.

BACKGROUND OF THE INVENTION

[0003] T cell activation is a serial process involving multiplesignaling pathways and sequential changes in gene expression resultingin differentiation of T cells into distinct subpopulations, i.e. Th1 andTh2, which are distinguishable by their pattern of cytokine productionand characterize the mode of the cellular immune response (Abbas et al.,1996; Crabtree, 1989). The T cell response is initiated by theinteraction of the antigen-specific T cell receptor (TCR) with peptidepresented by major histocompatibility complex (MHC) molecules on thesurface of antigen presenting cells (APCs). Additional signals areprovided by a network of receptor-ligand interactions mediated by anumber of membrane proteins such as CD28/CTLA4 and B7, CD40/CD40L, LFA-1and ICAM-1 (Lenschow et al., 1996; Linsley and Ledbetter, 1993; Xu etal., 1994, Bachmann et al., 1997; Schwartz, 1992), collectively calledcostimulatory signals (Perez et al., 1997). These membrane proteins canalter T cell activation in distinct ways (Bachmann et al., 1997) andregulate the immune response by the integration of positive and negativesignals provided by these molecules (Bluestone, 1995; Perez et al.,1997). Many of the agents which are effective in modulating the cellularimmune response either interfere with the T cell receptor (Cosimi etal., 1981), block costimulatory signaling (Larsen et al., 1996; Blazaret al., 1996; Kirk et al., 1997; Linsley et al., 1992; Turka et al.,1992) or inhibit intracellular activation signals downstream from theseprimary cell membrane triggers (Schreiber and Crabtree, 1992).Therapeutic prevention of T cell activation in organ transplantation andautoimmune diseases presently relies on panimmunosuppressive drugsinterfering with downstream intracellullar events. Specific modulationof the T cell response remains a longstanding goal in immunologicalresearch.

SUMMARY OF THE INVENTION

[0004] The present invention relates to polynucleotides encoding a novelT-cell membrane protein. Furthermore, the present invention relates topeptides and polypeptides derived therefrom as well as to antibodiescapable of inhibiting T-cell stimulation through the novel T-cellmembrane protein. More particulary, the present invention relates toapplications in the medical field that directly arise from thepolynucleotides, peptides, (poly)peptides and antibodies of theinvention. Additionally, the present invention relates to a novel methodfor testing activators and inhibitors of T-cell proliferation. Thepharmaceutical compositions, methods and uses of the invention areuseful therapeutically in situations where it is desirable to modulate(antigen-specific) immune responses, e.g., inducing and maintain(antigen-specific) T-cell unresponsiveness or restore (antigen-specific)T-cell responsiveness. For example, it may be necessary to induce ormaintain T-cell unresponsiveness in a subject who has received an organor bone marrow transplant to prevent graft rejection by inhibitingstimulation through the TIRC7 membrane protein. In addition, T-cellunresponsiveness can be maintained by blocking TIRC7 stimulation in asubject who has an autoimmune disease to alleviate symptoms of theautoimmune disease. In these cases, a TIRC7 inhibitory agent isadministered to the subject in an amount and over a period of timesufficient to maintain T-cell unresponsiveness. Alternatively, T-cellunresponsiveness can be reversed in a subject bearing a tumor tostimulate a tumor specific T-cell response or in a subject receiving avaccine to enhance the efficacy of the vaccine. For example, a cell(e.g., a tumor cell) can be modified to express a TIRC7 ligand or aTIRC7 stimulatory agent can be administered to the subject bearing atumor or who has had a tumor surgically removed to prevent recurrence ofthe tumor. Additionally, antigen-specific responsiveness can be restoredto anergized T-cells in vitro by stimulating the T-cells through TIRC7.Responsive T-cells generated in vitro can then be administered to asubject.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0005] In view of the need of therapeutic means for the treatment ofdiseases related to immune responses of the human body, the technicalproblem of the invention is to provide means and methods for themodulation of T-cell responses which are particularly useful in organtransplantation and autoimmune diseases.

[0006] The solution to this technical problem is achieved by providingthe embodiments characterized in the claims, namely a novel T-cellmembrane protein encoded by T-cell Immune Response cDNA7 (TIRC7) isdescribed which exhibits a central role in T-cell activation in vitroand in vivo. TIRC7 mRNA is transiently upregulated in the early phase ofT-cell activation via a calcineurin-dependent pathway.

[0007] In a first set of experiments, the TIRC7 protein encoding cDNAhas been cloned and characterized; see the first part of Example 1 andFIG. 1. Furthermore, said cDNA was subjected to in vitro translation andpreliminary results obtained in MLR inhibition tests with the in vitrotranslated TIRC7 protein suggested a putative immunosuppressivepotential of said protein, see Example 2 with FIG. 5B.

[0008] A second (independent) set of experiments performed in accordancewith the present invention (Examples 2 to 4), surprisingly revealed thatmodulation of TIRC7 membrane protein mediated signals with specificanti-TIRC7 membrane protein antibodies in vitro efficiently preventsT-cell proliferation and IL-2 secretion which is reversible by exogenousIL-2. Anti-TIRC7 membrane protein antibodies specifically inhibit Th1subset specific cytokine expression but spare the Th2 cytokines.Administration of said antibodies in rats significantly prolongs kidneyallograft survival. The latter described results obtained in accordancewith the present invention provide evidence for an essential role ofTIRC7 membrane protein in the early events of T cell activation. Thus,targeting of TIRC7 membrane protein and its encoding gene provides anovel therapeutic approach for modulation of the immune response.

[0009] Accordingly, the invention relates to a polynucleotide encoding aTIRC7 membrane protein or a biologically active fragmentthereofcomprising a DNA sequence selected from the group consisting of

[0010] (i) DNA sequences comprising a nucleotide sequence the amino acidsequence depicted in SEQ ID NO. 2 or SEQ ID NO. 13 from amino acidposition 1 to 614 or from amino acid position 1 to 601;

[0011] (ii) DNA sequences comprising the nucleotide sequence depicted inSEQ ID NO. 1 or SEQ ID NO. 12;

[0012] (iii) DNA sequences comprising a nucleotide sequence encoding afragment or derivative of the protein encoded by the DNA sequence of (i)or (ii); and

[0013] (iv) DNA sequences the complementary strand of which hybridizeswith and which is at least 70% identical to the polynucleotide asdefined in any one of (i) to (iii).

[0014] The term “TIRC7 membrane protein” as used in accordance with thepresent invention, denotes a protein involved in the signal transductionof T-cell activation and/or proliferation and that, preferably in asoluble form is capable of inhibiting or suppressing T-cellproliferation in response to alloactivation in a mixed lymphocyteculture or in response to mitogens when exogeneously added to theculture. Studies which had been carried out within the scope of thepresent invention revealed that soluble in vitro translated TIRC7protein is able to efficiently suppress in a dose dependent manner theproliferation of T-cells in response to alloactivation in a mixedlymphocyte culture or in response to mitogens; see Example 2, FIG. 5B.

[0015] The term “biologically active fragment thereof” refers topeptides and polypeptides that are derived from said TIRC7 membraneprotein and that are capable of inhibiting T-cell proliferation asdefined above.

[0016] Previously, a cDNA fragment was amplified by the ReverseTranscription Differential Display Polymerase Chain Reaction (DDRT-PCR)technique from RNA from cells stimulated in mixed lymphocyte culture(MLC). The amino acid sequence of the 350 base-pair (bp) cDNA fragmentthus obtained was reported to belong to a new human (H+)-ATPase protonpump homologue differentially expressed in alloactivated lymphocytes asthe amino acid sequence had substantial homology to a rat and bovinevesicular (H+)-ATPase proton pump, respectively. While the function ofthe molecule was not known, it was also speculated that its homology toa mouse T-cell derived immunosuppressive protein (J6B7, Lee, MolecularImmunology 27 (1990), 1137-1144) suggested that the corresponding genemay be involved in immunomodulation. A full-length cDNA clone wasisolated by means of the 350 base-pair cDNA fragment from a library ofactivated human T-cells. However, the amino acid sequence of the newlycloned cDNA did not reveal further insights into the putative functionof the encoded protein although some initial experiments concerning theexpression pattern of gene supposedly corresponding to the 350 bpfragment and its encoded, in vitro translated protein were reported andgave rise to speculation. Furthermore, the attempt to clone the mousehomologue with the help of either the 350 bp fragment or the full-lengthcDNA failed.

[0017] Further investigations revealed, that the amino acid sequence ofthe cDNA fragment identified by DDRT-PCR from RNA of stimulatedT-lymphocytes corresponds to a transmembrane region. Thus, it could notbe ruled out that due to conserved structures and amino acid sequencesof such transmembrane regions the full-length cDNA clone did notactually correspond to the fragment identified by DDRT-PCR. For example,there are 5 amino acid differences in the amino acid sequence of theTIRC7 protein compared with that of the transmembrane region encoded bythe 350 bp fragment. Furthermore, since DDRT-PCR analysis of RNA fromhuman lymphocytes identified several genes whose expression was changedwith alloactivation (multi) gene families may exist of similar structureand/or function while only one or few members of those genes areactually identified by the above-described technique. Hence, therelevance of the preliminary results described above and the actualbiological function of this unknown protein being without precedent wereabsolutely unclear.

[0018] The preliminary results were all the more questioned in view ofthe recent publication of Lee, Biochem. Biophys. Res. Communications 218(1996), 813-821, wherein the cloning and characterization of a humanosteoclast-specific 116 kDa proton pump subunit was reported the cDNA ofwhich comprises a nucleotide and an amino acid sequence which issubstantially identical to those of the gene cloned from activated humanT-cells. Thus, it appeared as if the previous results reflected anartefact and/or were obtained due to the activity of a pseudo-gene ofthe gene described in Lee, supra.

[0019] In accordance with the present invention a polynucleotide withthe nucleotide sequence of the coding region as depicted in SEQ ID NO: 1has been identified encoding a protein of 614 amino acids (SEQ ID NO: 2)with a molecular weight of 75 kDA. Experiments performed in accordancewith the present invention revealed that TIRC7 membrane protein isexpressed in all lymphoid tissues with low expression only in thymus,bone marrow and fetal liver and is transiently up regulated inlymphocytes after stimulation of the T-cell receptor, see Example 1,FIG. 3. The TIRC7 encoding gene has been located by using thefluorescence-in situ-hybridization (FISH) method on the long arm ofhuman chromosome 10 (13.4-13.5q) which is close to the breakpoint regionof the bcl-gene associated with leukemia. The TIRC7 membrane protein ispredominantly expressed on the cell membrane, consistent with a targetfor an external ligand; see Example 1, FIG. 4. The seven transmembranedomain structure predicts three extracellular loops and anextracellularly oriented carboxy terminus; see FIG. 2.

[0020] From the above it is evident that the nucleotide sequencedepicted in SEQ ID No. 1 encodes a novel class of T-cell membraneproteins. By the provision of this nucleotide sequence it is nowpossible to isolate identical or similar polynucleotides which code forproteins with the biological activity of TIRC7 from other species ororganisms. Well-established approaches for the identification andisolation of such related sequences are, for example, the isolation fromgenomic or cDNA libraries using the complete or part of the disclosedsequence as a probe or the amplification of correspondingpolynucleotides by polymerase chain reaction using specific primers. Inaccordance with the present invention, a further polynucleotide encodinga TIRC7 membrane protein was isolated using a nucleic acid moleculecomprising the coding sequence of SEQ ID NO: 1 as a probe. Thenucleotide sequence of said polynucleotide is given in SEQ ID NO: 12encoding a protein having the amino acid sequence of SEQ ID NO: 13. Thenucleotide and amino acid sequences of said TIRC7 membrane protein aresubstantially identical with those of the TIRC7 membrane protein encodedby SEQ ID NO: 1 except at amino acid position 121 (Arg® Gln) and,therefore, presumably represent allelic variants.

[0021] Thus, the invention also relates to polynucleotides whichhybridize to the above described polynucleotides and differ at one ormore positions in comparison to these as long as they encode a TIRC7membrane protein as defined above. Such molecules comprise those whichare changed, for example, by deletion(s), insertion(s), alteration(s) orany other modification known in the art in comparison to the abovedescribed polynucleotides either alone or in combination. Methods forintroducing such modifications in the polynucleotides of the inventionare well-known to the person skilled in the art; see, e.g., Sambrook etal. (Molecular cloning; A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor N.Y. (1989)). The inventionalso relates to polynucleotides the nucleotide sequence of which differsfrom the nucleotide sequence of any of the above-describedpolynucleotides due to the degeneracy of the genetic code.

[0022] With respect to the DNA sequences characterized under (iv) above,the term “hybridizing” in this context is understood as referring toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37° C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55° C. Most preferably, the term“hybridizing” refers to stringent hybridization conditions, for examplesuch as described in Sambrook, supra.

[0023] Particularly preferred are polynucleotides which share 70%,preferably at least 85%, more preferably 90-95%, and most preferably96-99% sequence identity with one of the above-mentioned polynucleotidesand have the same biological activity. Such polynucleotides alsocomprise those which are altered, for example by nucleotide deletion(s),insertion(s), substitution(s), addition(s), and/or recombination(s)and/or any other modification(s) known in the art either alone or incombination in comparison to the above-described polynucleotides.Methods for introducing such modifications in the nucleotide sequence ofthe polynucleotide of the invention are well known to the person skilledin the art. Thus, the present invention encompasses any polynucleotidethat can be derived from the above described polynucleotides by way ofgenetic engineering and that encode upon expression a TIRC7 membraneprotein or a biologically active fragment thereof.

[0024] It is also immediately evident to the person skilled in the artthat regulatory sequences may be added to the polynucleotide of theinvention. For example, promoters, transcriptional enhancers and/orsequences which allow for induced expression of the polynucleotide ofthe invention may be employed. A suitable inducible system is forexample tetracycline-regulated gene expression as described, e.g., byGossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551) andGossen et al. (Trends Biotech. 12 (1994), 58-62).

[0025] In a further embodiment, the invention relates to nucleic acidmolecules of at least 15 nucleotides in length hybridizing specificallywith a polynucleotide as described above or with a complementary strandthereof. Specific hybridization occurs preferably under stringentconditions and implies no or very little cross-hybridization withnucleotide sequences encoding no or substantially different proteins.Such nucleic acid molecules may be used as probes and/or for the controlof gene expression. Nucleic acid probe technology is well known to thoseskilled in the art who will readily appreciate that such probes may varyin length. Preferred are nucleic acid probes of 17 to 35 nucleotides inlength. Of course, it may also be appropriate to use nucleic acids of upto 100 and more nucleotides in length. The nucleic acid probes of theinvention are useful for various applications. On the one hand, they maybe used as PCR primers for amplification of polynucleotides according tothe invention. Another application is the use as a hybridization probeto identify polynucleotides hybridizing to the polynucleotides of theinvention by homology screening of genomic DNA libraries. Nucleic acidmolecules according to this preferred embodiment of the invention whichare complementary to a polynucleotide as described above may also beused for repression of expression of a gene comprising such apolynucleotide, for example due to an antisense or triple helix effector for the construction of appropriate ribozymes (see, e.g., EP-B1 0 291533, EP-A1 0 321 201, EP-A2 0 360 257) which specifically cleave the(pre)-mRNA of a gene comprising a polynucleotide of the invention.Selection of appropriate target sites and corresponding ribozymes can bedone as described for example in Steinecke, Ribozymes, Methods in CellBiology 50, Galbraith et al. eds Academic Press, Inc. (1995), 449-460.Standard methods relating to antisense technology have also beendescribed (Melani, Cancer Res. 51 (1991), 2897-2901). Furthermore, theperson skilled in the art is well aware that it is also possible tolabel such a nucleic acid probe with an appropriate marker for specificapplications, such as for the detection of the presence of apolynucleotide of the invention in a sample derived from an organism.

[0026] The above described nucleic acid molecules may either be DNA orRNA or a hybrid thereof. Furthermore, said nucleic acid molecule maycontain, for example, thioester bonds and/or nucleotide analogues,commonly used in oligonucleotide anti-sense approaches. Saidmodifications may be useful for the stabilization of the nucleic acidmolecule against endo- and/or exonucleases in the cell. Said nucleicacid molecules may be transcribed by an appropriate vector containing achimeric gene which allows for the transcription of said nucleic acidmolecule in the cell. Such nucleic acid molecules may further containribozyme sequences as described above.

[0027] In this respect, it is also to be understood that thepolynucleotide of the invention can be used for “gene targeting” and/or“gene replacement”, for restoring a mutant gene or for creating a mutantgene via homologous recombination; see for example Mouellic, Proc. Natl.Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, APractical Approach, Oxford University Press.

[0028] In a preferred embodiment said nucleic acid molecules arelabeled. Methods for the detection of nucleic acids are well known inthe art, e.g., Southern and northern blotting, PCR or primer extension.In another preferred embodiment said nucleic acid molecules may be usedfor the suppression of TIRC7 expression.

[0029] The polynucleotide of the invention encoding the above describedTIRC7 membrane protein or biologically active fragments thereof may be,e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or arecombinantly produced chimeric nucleic acid molecule comprising any ofthose polynucleotides either alone or in combination. Preferably saidpolynucleotide is part of a vector. Such vectors may comprise furthergenes such as marker genes which allow for the selection of said vectorin a suitable host cell and under suitable conditions. Preferably, thepolynucleotide of the invention is operatively linked to expressioncontrol sequences allowing expression in prokaryotic or eukaryoticcells. Expression of said polynucleotide comprises transcription of thepolynucleotide into a translatable mRNA. Regulatory elements ensuringexpression in eukaryotic cells, preferably mammalian cells, are wellknown to those skilled in the art. They usually comprise regulatorysequences ensuring initiation of transcription and optionally poly-Asignals ensuring termination of transcription and stabilization of thetranscript. Additional regulatory elements may include transcriptionalas well as translational enhancers, and/or naturally-associated orheterologous promoter regions. Possible regulatory elements permittingexpression in prokaryotic host cells comprise, e.g., the P_(L), lac, trpor tac promoter in E. coli, and examples for regulatory elementspermitting expression in eukaryotic host cells are the AOX1 or GAL1promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus),CMV-enhancer, SV40-enhancer or a globin intron in mammalian and otheranimal cells. Beside elements which are responsible for the initiationof transcription such regulatory elements may also comprisetranscription termination signals, such as the SV40-poly-A site or thetk-poly-A site, downstream of the polynucleotide. Furthermore, dependingon the expression system used leader sequences capable of directing thepolypeptide to a cellular compartment or secreting it into the mediummay be added to the coding sequence of the polynucleotide of theinvention and are well known in the art. The leader sequence(s) is (are)assembled in appropriate phase with translation, initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein, or a portion thereof, intothe periplasmic space or extracellular medium. Optionally, theheterologous sequence can encode a fusion protein including an C- orN-terminal identification peptide imparting desired characteristics,e.g., stabilization or simplified purification of expressed recombinantproduct. In this context, suitable expression vectors are known in theart such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia),pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), or pSPORT1 (GIBCO BRL).

[0030] Preferably, the expression control sequences will be eukaryoticpromoter systems in vectors capable of transforming or transfectingeukaryotic host cells, but control sequences for prokaryotic hosts mayalso be used. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and, as desired, the collectionand purification of the protein of the invention may follow; see, e.g.,the appended examples.

[0031] In accordance with the above, the present invention relates tovectors, particularly plasmids, cosmids, viruses and bacteriophages usedconventionally in genetic engineering that comprise a polynucleotide ofthe invention. Methods which are well known to those skilled in the artcan be used to construct recombinant vectors; see, for example, thetechniques described in Sambrook, Molecular Cloning A Laboratory Manual,Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocolsin Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y. (1989). Alternatively, the polynucleotides andvectors of the invention can be reconstituted into liposomes fordelivery to target cells. The vectors containing the polynucleotides ofthe invention can be transferred into the host cell by well-knownmethods, which vary depending on the type of cellular host. For example,calcium chloride transfection is commonly utilized for prokaryoticcells, whereas calcium phosphate treatment or electroporation may beused for other cellular hosts; see Sambrook, supra.

[0032] In a still further embodiment, the present invention relates to acell containing the polynucleotide or vector described above.Preferably, said cell is a eukaryotic, most preferably a mammalian cellif therapeutic uses of the protein are envisaged. Of course, yeast andless preferred prokaryotic, e.g., bacterial cells may serve as well, inparticular if the produced protein is used as a diagnostic means.

[0033] The polynucleotide or vector of the invention which is present inthe host cell may either be integrated into the genome of the host cellor it may be maintained extrachromosomally.

[0034] The term “prokaryotic” is meant to include all bacteria which canbe transformed or transfected with a DNA or RNA molecules for theexpression of a protein of the invention. Prokaryotic hosts may includegram negative as well as gram positive bacteria such as, for example, E.coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. Theterm “eukaryotic” is meant to include yeast, higher plant, insect andpreferably mammalian cells. Depending upon the host employed in arecombinant production procedure, the protein encoded by thepolynucleotide of the present invention may be glycosylated or may benon-glycosylated. TIRC7 proteins of the invention may also include aninitial methionine amino acid residue. A polynucleotide of the inventioncan be used to transform or transfect the host using any of thetechniques commonly known to those of ordinary skill in the art.Furthermore, methods for preparing fused, operably linked genes andexpressing them in, e.g., mammalian cells and bacteria are well-known inthe art (Sambrook, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989). The geneticconstructs and methods described therein can be utilized for expressionof the TIRC7 protein of the invention in eukaryotic or prokaryotichosts. In general, expression vectors containing promoter sequenceswhich facilitate the efficient transcription of the insertedpolynucleotide are used in connection with the host. The expressionvector typically contains an origin of replication, a promoter, and aterminator, as well as specific genes which are capable of providingphenotypic selection of the transformed cells. Furthermore, transgenicanimals, preferably mammals, comprising cells of the invention may beused for the large scale production of the TIRC7 protein of theinvention.

[0035] Alternatively, an animal, preferably mammalian cell naturallyhaving a polynucleotide of the invention present in its genome can beused and modified such that said cell expresses the endogenus genecorresponding to the polynucleotide of the invention under the controlof an heterologous promoter. The introduction of the heterologouspromoter which does not naturally control the expression of thepolynucleotide of the invention can be done according to standardmethods, see supra. Suitable promoter include those mentionedhereinbefore.

[0036] Thus, in a further embodiment, the present invention relates to amethod for the production of a TIRC7 membrane protein or a biologicallyactive fragment thereof comprising:

[0037] (a) culturing a host of the invention under conditions allowingfor the expression of the protein; or

[0038] (b) in vitro translation of the polynucleotide of the invention;

[0039] and recovering the protein produced in (a) or (b).

[0040] The transformed hosts can be grown in fermentors and culturedaccording to techniques known in the art to achieve optimal cell growth.The TIRC7 protein of the invention can then be isolated from the growthmedium, cellular lysates, or cellular membrane fractions. Onceexpressed, the protein of the present invention can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like; see, Scopes, “Protein Purification”,Springer-Verlag, N.Y. (1982). Substantially pure proteins of at leastabout 90 to 95% homogeneity are preferred, and 98 to 99% or morehomogeneity are most preferred, for pharmaceutical uses. Once purified,partially or to homogeneity as desired, the proteins may then be usedtherapeutically (including extracorporeally) or in developing andperforming assay procedures.

[0041] Hence, in a still further embodiment, the present inventionrelates to a TIRC7 membrane protein or a biologically active fragmentthereof encoded by the polynucleotide of the invention or produced by amethod of as described above. It will be apparent to those skilled inthe art that the protein of the invention can be further coupled toother moieties as described above for, e.g., drug targeting and imagingapplications. Such coupling may be conducted chemically after expressionof the protein to site of attachment or the coupling product may beengineered into the protein of the invention at the DNA level. The DNAsare then expressed in a suitable host system, and the expressed proteinsare collected and renatured, if necessary. Furthermore, the provision ofthe TIRC7 protein of the present invention enables the production ofTIRC7 specific antibodies. In this respect, hybridoma technology enablesproduction of cell lines secreting antibody to essentially any desiredsubstance that produces an immune response. RNA encoding the light andheavy chains of the immunoglobulin can then be obtained from thecytoplasm of the hybridoma. The 5′ end portion of the mRNA can be usedto prepare cDNA to be inserted into an expression vector. The DNAencoding the antibody or its immunoglobulin chains can subsequently beexpressed in cells, preferably mammalian cells.

[0042] Depending on the host cell, renaturation techniques may berequired to attain proper conformation of the antibody. If necessary,point substitutions seeking to optimize binding may be made in the DNAusing conventional cassette mutagenesis or other protein engineeringmethodology such as is disclosed herein.

[0043] Thus, the present invention also relates to an antibodyspecifically recognizing the peptide or polypeptide of the invention.

[0044] In a still further embodiment, the present invention relates to acell that has been modified to express a TIRC7 protein or an antibody ofthe invention. This embodiment may be well suited for, e.g., restoringT-cell responsiveness to an antigen, in particular if the antibody ofthe invention capable of stimulating T-cell proliferation is expressedin a form suitable to be presented on the cell surface.

[0045] Moreover, the present invention relates to pharmaceuticalcompositions comprising a peptide or polypeptide being capable ofinhibiting T-cell stimulation through the TIRC7 membrane protein and/orbeing recognized by an antibody capable of inhibiting T-cell stimulationthrough the TIRC7 membrane protein encoded by a fragment of the abovedescribed polynucleotides or an antibody specifically recognizing saidpeptide or polypeptide. The term “capable of inhibiting T-cellstimulation through TIRC7 membrane protein” denotes the ability ofsuppressing the proliferation of T-cells in response to alloactivationin a mixed lymphocyte culture or in response to mitogens by way ofblocking or antagonizing the biological activity of the TIRC7 membraneprotein herein also referred to as TIRC7 protein or TIRC7. The terms“capable of inhibiting T-cell stimulation through TIRC7 membraneprotein” and “inhibiting TIRC7 activity” are used interchangeableherein.

[0046] Studies which had been carried out within the scope of thepresent invention surprisingly revealed that anti-TIRC7 antibodiesdirected against the extracellular domains, but not those recognizingpredicted intracellular domains of the protein, are able to efficientlysuppress the proliferation of T-cells in response to alloactivation in amixed lymphocyte culture or in response to mitogens; see Example 2, FIG.5. Similar results were obtained with in vitro translated soluble TIRC7protein. The inhibitory effect of anti-TIRC7 antibodies on T-cellsinduced by a variety of different stimulatory pathways suggests thatTIRC7 plays a central role in T cell activation. Moreover, inhibition ofT cell proliferation in MLR by antibody targeting of TIRC7 suggest theexistence of a ligand specifically interacting with TIRC7. Support forthis hypothesis is provided by the dose-dependent inhibition of T cellproliferation in a MLR in the presence of soluble in vitro translatedTIRC7 protein.

[0047] Furthermore, it could be shown in accordance with the presentinvention that antibody targeting of TIRC7 has a selective inhibitoryeffect on the Th1 lymphocyte subset, as evidenced by the inhibition ofIL-2 and IFN-g, but not IL-4, cytokine production; see Example 3, FIG.5. With anti-TIRC7 antibody treatment the cells appear to remain in anunresponsive, but functional, state since exogenous recombinant IL-2reversed the antiproliferative effect of the anti-TIRC7 antibodies.

[0048] While the above described results hold promise that the novelTIRC7 protein, and antibodies thereto may be therapeutically useful,proof for the concept of the invention, namely the usefulness of theabove described-compounds for the modulation of the immune response aswell as the embodiments derived therefrom and characterized hereinbelowcame from further experiments performed in accordance with the presentinvention demonstrating the ability of an anti-TIRC7 antibody to preventallograft rejection in the in vivo model of rat kidney transplantation;see Example 4, FIGS. 6 und 7. Moreover, it could be demonstrated inaccordance with the present invention that advantageously the effects ofantibody targeting of TIRC7 are quite similar to those observed bytargeting of costimulatory molecules. Antibody blocking of costimulatorymolecules has been shown to inhibit human T cell proliferation (Linsleyet al., 1992; Walunas et al., 1994). Furthermore, interruption ofCD28/B7 interaction with the soluble protein CTLA4Ig caused inhibitionof T cell proliferation (Linsley et al., 1992; Lenschow et al., 1992;Larsen et al., 1996). Further analogy to the effect of TIRC7 antibodytargeting is provided by CTLA4Ig selectively blocking Th1 and sparingTh2 lymphocyte responses (Mohammed et al., 1995). It was shown, thatadministration of CTLA4Ig in an in vivo model of kidney allografttransplantation prolonged graft survival (Mohammed et al., 1995) whichwas similarly observed by TIRC7 antibody targeting in the presentExamples. Although these similarities may suggest a costimulatoryfunction, TIRC7 does not share structural or sequence homology with anyof the known T cell accessory molecules. Thus, TIRC7 may participitatein a distinct signaling pathway induced early in the course of T cellactivation. This possibility is supported by recent reports thatinterference with pathways mediated by molecules other than the knowncostimulatory proteins can modulate the T cell response. For example,antibody targeting of the common leukocyte antigen CD45RB was shown toresult in a prevention of graft rejection in mice (Lazarovits et al.,1996). Given the functional similarities between TIRC7 and the known Tcell accessory molecules, it is expected that the structural novelty ofTIRC7 will contribute to the understanding of distinct mechanisms in theT cell response. Moreover, the striking capacity of anti-TIRC7 antibodyto significantly prolong allograft survival in vivo provide a novelapproach for a selective inhibition of undesired T cell activation inhuman organ transplantation and autoimmune diseases.

[0049] In a preferred embodiment of the invention, said peptide orpolypeptide encoded by the above described polynucleotide comprises theamino acid sequence depicted in any one of SEQ ID NOS 3 to 9. As isdescribed in the appended examples peptides comprising the abovementioned amino acid sequences correspond to parts of the extracellulardomain of the TIRC7 protein and can advantageously be used for thegeneration of antibodies that are capable of inducing T-cellunresponsiveness.

[0050] Accordingly, in a particularly preferred embodiment thepharmaceutical composition of the invention comprises a soluble form ofsaid peptide or polypeptide. In one embodiment, a soluble form of TIRC7or a TIRC7 ligand is a truncated form of the molecule comprising anextracellular domain of the TIRC7 or a functional portion thereof. Aportion of the extracellular domain of TIRC7 which retains the abilityto bind to a TIRC7 ligand can be used. Likewise, a portion of theextracellular domain of a TIRC7 ligand which retains the ability to bindto TIRC7 can be used. Another soluble form of TIRC7 or a TIRC7 ligandfor use in accordance with the present invention is a fusion protein.The term “fusion protein” as used herein refers to a protein comprisedof a first polypeptide from a first protein in contiguous amino acidsequence with a second polypeptide from a second protein. Fusionproteins can be made by standard recombinant DNA techniques wherein anucleotide sequence encoding the first polypeptide is ligated in-frameto a nucleotide sequence encoding the second polypeptide, and thesenucleotide sequences are expressed (e.g., using a recombinant expressionvector introduced into a host cell) to produce the fusion protein. Apreferred fusion protein is an immunoglobulin fusion protein whichincludes an extracellular domain, or functional portion of TIRC7 or aTIRC7 ligand linked to an immunoglobulin heavy chain constant region(e.g., the hinge, CH2 and CH3 regions of a human immunoglobulin such asIgG1). Immunoglobulin fusion proteins can be prepared, for example,according to the teachings of Capon, Nature 337 (1989), 525-531.

[0051] The antibody comprised in the pharmaceutical composition of theinvention preferably has a specificity at least substantially identicalto the binding specificity of the natural ligand of the TIRC7 protein ofthe invention, in particular if T-cell stimulation is desired. Such anantibody can have a binding affinity of at least 10⁵M⁻¹, preferably nothigher than 10⁷M⁻¹ if T-cell stimulation is envisaged and advantageouslyup to 10¹⁰M⁻¹ in case T-cell suppression should be mediated.

[0052] In a preferred embodiment, antibody

[0053] (a) a T-cell suppressive antibody has an affinity of at leastabout 10⁻⁷ M, preferably at least about 10⁻⁹ M and most preferably atleast about 10−11 M; and

[0054] (b) a T-cell stimulating antibody has an affinity of less thanabout 10⁻⁷ M, preferably less than about 10⁻⁶ M and most preferably inorder of 10⁻⁵M.

[0055] In case of bispecific antibodies where one specificity isdirected to a target cell to be destroyed, e.g. a tumor cell, it isadvantageous if the binding site recognizing the tumor antigen has ahigh affinity in order to capture the target cells to be destroyed withhigh efficiency. On the other hand, the binding affinity of the bindingsite recognizing the TIRC7 protein of the invention should be in theorder of those of the natural TIRC7 ligand or of that usually found forthe interaction of the T-cell costimulatory molecules with theirreceptor.

[0056] In a preferred embodiment of the invention, said antibody is amonoclonal antibody, a polyclonal antibody, a single chain antibody,humanized antibody, or fragment thereof that specifically binds saidpeptide or polypeptide also including bispecific antibody, syntheticantibody, antibody fragment, such as Fab, Fv or scFv fragments etc., ora chemically modified derivative of any of these. Monoclonal antibodiescan be prepared, for example, by the techniques as originally describedin Kohler and Milstein, Nature 256 (1975), 495, and Galfré, Meth.Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cellsto spleen cells derived from immunized mammals with modificationsdeveloped by the art. Furthermore, antibodies or fragments thereof tothe aforementioned peptides can be obtained by using methods which aredescribed, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”,CSH Press, Cold Spring Harbor, 1988. When derivatives of said antibodiesare obtained by the phage display technique, surface plasmon resonanceas employed in the BIAcore system can be used to increase the efficiencyof phage antibodies which bind to an epitope of the peptide orpolypeptide of the invention (Schier, Human Antibodies Hybridomas 7(1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). Theproduction of chimeric antibodies is described, for example, inWO89/09622. Methods for the production of humanized antibodies aredescribed in, e.g., EP-A1 0 239 400 and WO90/07861. A further source ofantibodies to be utilized in accordance with the present invention areso-called xenogenic antibodies. The general principle for the productionof xenogenic antibodies such as human antibodies in mice is describedin, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735.

[0057] Antibodies to be employed in accordance with the invention ortheir corresponding immunoglobulin chain(s) can be further modifiedusing conventional techniques known in the art, for example, by usingamino acid deletion(s), insertion(s), substitution(s), addition(s),and/or recombination(s) and/or any other modification(s) known in theart either alone or in combination. Methods for introducing suchmodifications in the DNA sequence underlying the amino acid sequence ofan immunoglobulin chain are well known to the person skilled in the art;see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory (1989) N.Y.

[0058] The peptides, polypeptides and antibodies comprised in thepharmaceutical compositions of the present invention can comprise afurther domain, said domain being linked by covalent or non-covalentbonds. The linkage can be based on genetic fusion according to themethods known in the art and described above or can be performed by,e.g., chemical cross-linking as described in, e.g., WO 94/04686. Theadditional domain present in the fusion protein comprising the peptide,polypeptide or antibody employed in accordance with the invention maypreferably be linked by a flexible linker, advantageously a polypeptidelinker, wherein said polypeptide linker comprises plural, hydrophilic,peptide-bonded amino acids of a length sufficient to span the distancebetween the C-terminal end of said further domain and the N-terminal endof the peptide, polypeptide or antibody or vice versa. The abovedescribed fusion protein may further comprise a cleavable linker orcleavage site for proteinases.

[0059] Furthermore, said further domain may be of a predefinedspecificity or function. For example, the literature contains a host ofreferences to the concept of targeting bioactive substances such asdrugs, toxins, and enzymes to specific points in the body to destroy orlocate malignant cells or to induce a localized drug or enzymaticeffect. It has been proposed to achieve this effect by conjugating thebioactive substance to monoclonal antibodies (see, e.g., N.Y. OxfordUniversity Press; and Ghose, (1978) J. Natl. Cancer Inst. 61:657-676).

[0060] In this context, it is understood that the polypeptides presentin the pharmaceutical composition according to the invention may befurther modified by conventional methods known in the art. This allowsfor the construction of fusion proteins comprising the peptide,polypeptide or antibody of the invention and other functional amino acidsequences, e.g., nuclear localization signals, transactivating domains,DNA-binding domains, hormone-binding domains, protein tags (GST, GFP,h-myc peptide, Flag, HA peptide) which may be derived from heterologousproteins. Thus, administration of the composition of the invention canutilize unlabeled as well as labeled (poly)peptides or antibodies.

[0061] For example, the peptides, polypeptides and antibodies can beadministered labeled with a therapeutic agent. These agents can becoupled either directly or indirectly to the antibodies or(poly)peptides of the invention, see supra, and can be selected toenable drug release from the antigen at the target site. Examples oftherapeutic agents which can be coupled to the (poly)peptides andantibodies for immunotherapy are drugs, radioisotopes, lectins, andtoxins. The drugs which can be conjugated to the polypeptides of theinvention include compounds which are classically referred to as drugssuch as mitomycin C, daunorubicin, and vinblastine.

[0062] In using radioisotopically conjugated (poly)peptides orantibodies of the invention for, e.g., immunotherapy, certain isotopesmay be more preferable than others depending on such factors asleukocyte distribution as well as stability and emission. Depending onthe autoimmune response, some emitters may be preferable to others. Ingeneral, a and b particle-emitting radioisotopes are preferred inimmunotherapy. Preferred are short range, high energy a emitters such as²¹²Bi. Examples of radioisotopes which can be bound to the(poly)peptides and antibodies of the invention for therapeutic purposesare ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹²Bi, ²¹²At, ²¹¹Pb, ⁴⁷Sc, ¹⁰⁹Pd and ¹⁸⁸Re.

[0063] Lectins are proteins, usually isolated from plant material, whichbind to specific sugar moieties. Many lectins are also able toagglutinate cells and stimulate lymphocytes. However, ricin is a toxiclectin which has been used immunotherapeutically. This is accomplishedby binding the a-peptide chain of ricin, which is responsible fortoxicity, to the polypeptide to enable site specific delivery of thetoxic effect.

[0064] Toxins are poisonous substances produced by plants, animals, ormicroorganisms that, in sufficient dose, are often lethal. Diphtheriatoxin is a substance produced by Corynebacterium diphtheria which can beused therapeutically. This toxin consists of an a and b subunit whichunder proper conditions can be separated. The toxic A component can bebound to an antibody of the invention and be used for site specificdelivery to the interacting T-cell.

[0065] Other therapeutic agents such as described above which can becoupled to the polypeptide of the invention, as well as correspoding exvivo and in vivo therapeutic protocols, are known, or can be easilyascertained, by those of ordinary skill in the art. Wherever appropriatethe person skilled in the art may use a polynucleotide of the inventiondescribed hereinbelow encoding any one of the above described(poly)peptides and antibodies, respectively, or the correspondingvectors instead of the proteinaeous material itself.

[0066] Thus, the person skilled in the art will readily appreciate thatthe (poly)peptide and antibody of the invention can be used for theconstruction of fusion proteins of desired specificity and biologicalfunction. The (poly)peptides and antibodies then optionally employed inaccordance with the present invention of the invention as well as fusionprotein thereof are expected to play an important therapeutic andscientific role in particular in the medical field, for example, in thedevelopment of new treatment approaches for T-cell related disorderssuch as certain forms of cancer and autoimmune diseases or asinteresting tools for the analysis and modulation of the correspondingcellular signal transduction pathways.

[0067] Preferably said further domain comprises a molecule selected fromthe group consisting of effector molecules having a conformationsuitable for biological activity, amino acid sequences capable ofsequestering an ion, and amino acid sequences capable of selectivebinding to a solid support or to a preselected antigen. Said domain maycomprises an enzyme, toxin, receptor, binding site, biosyntheticantibody binding site, growth factor, cell-differentiation factor,lymphokine, cytokine, hormone, a remotely detectable moiety,anti-metabolite, a radioactive atom or an antigen. Said antigen can be,e.g., tumor antigen, a viral antigen, a microbial antigen, an allergen,an auto-antigen, a virus, a microorganism, a polypeptide, a peptide or aplurality of tumor cells. Furthermore, said sequence capable ofsequestering an ion includes calmodulin, methallothionein, a fragmentthereof, or an amino acid sequence rich in at least one of glutamicacid, aspartic acid, lysine, and arginine. In addition, said polypeptidesequence capable of selective binding to a solid support can be apositively or negatively charged amino acid sequence, acysteine-containing amino acid sequence, avidin, streptavidin, afragment of Staphylococcus protein A, GST, a His-tag, a FLAG-tag, Lex Aor c-myc as used in the appended examples. Some of the effectormolecules and amino acid sequences described above may be present in aproform which itself is either active or not and which may be removed,when, e.g., entering a certain cellular environment.

[0068] The pharmaceutical composition of the present invention mayfurther comprise a pharmaceutically acceptable carrier. Examples ofsuitable pharmaceutical carriers are well known in the art and includephosphate buffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.Compositions comprising such carriers can be formulated by well knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose. Administration of thesuitable compositions may be effected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, topical orintradermal administration. The dosage regimen will be determined by theattending physician and clinical factors. As is well known in themedical arts, dosages for any one patient depends upon many factors,including the patient's size, body surface area, age, the particularcompound to be administered, sex, time and route of administration,general health, and other drugs being administered concurrently. Atypical dose can be, for example, in the range of 0.001 to 1000 μg (orof nucleic acid for expression or for inhibition of expression in thisrange); however, doses below or above this exemplary range areenvisioned, especially considering the aforementioned factors.Generally, the regimen as a regular administration of the pharmaceuticalcomposition should be in the range of 1 μg to 10 mg units per day. Ifthe regimen is a continuous infusion, it should also be in the range of1 μg to 10 mg units per kilogram of body weight per minute,respectively. Progress can be monitored by periodic assessment. Dosageswill vary but a preferred dosage for intravenous administration of DNAis from approximately 10⁶ to 10¹² copies of the DNA molecule. Thecompositions of the invention may be administered locally orsystemically. Administration will generally be parenterally, e.g.,intravenously; DNA may also be administered directly to the target site,e.g., by biolistic delivery to an internal or external target site or bycatheter to a site in an artery. Preparations for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like. Furthermore, thepharmaceutical composition of the invention may comprise further agentssuch as T-cell costimulatory molecules or cytokines known in the art, ortheir inhibitors or activators depending on the intended use of thepharmaceutical composition.

[0069] Furthermore, it is envisaged by the present invention that thevarious polynucleotides and vectors encoding the above describedpeptides or polypeptides are administered either alone or in anycombination using standard vectors and/or gene delivery systems, andoptionally together with a pharmaceutically acceptable carrier orexcipient. For example, the polynucleotide of the invention can be usedalone or as part of a vector to express the (poly)peptide of theinvention in cells, for, e.g., gene therapy or diagnostics of diseasesrelated to T-cell disorders. The polynucleotides or vectors of theinvention are introduced into the cells which in turn produce the(poly)peptide. Subsequent to administration, said polynucleotides orvectors may be stably integrated into the genome of the subject. On theother hand, viral vectors may be used which are specific for certaincells or tissues and persist in said cells. Suitable pharmaceuticalcarriers and excipients are well known in the art. The pharmaceuticalcompositions prepared according to the invention can be used for theprevention or treatment or delaying of different kinds of diseases,which are related to T-cell related immunodeficiencies and malignancies.

[0070] Furthermore, it is possible to use a pharmaceutical compositionof the invention which comprises polynucleotide or vector of theinvention in gene therapy. Gene therapy, which is based on introducingtherapeutic genes into cells by ex-vivo or in-vivo techniques is one ofthe most important applications of gene transfer. Suitable vectors andmethods for in-vitro or in-vivo gene therapy are described in theliterature and are known to the person skilled in the art; see, e.g.,Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79(1996), 911-919; Anderson, Science 256 (1992), 808-813; Isner, Lancet348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang,Nature Medicine 2 (1996), 714-716; WO94/29469; WO 97/00957 or Schaper,Current Opinion in Biotechnology 7 (1996) , 635-640, and referencescited therein. The polynucleotides and vectors of the invention may bedesigned for direct introduction or for introduction via liposomes, orviral vectors (e.g. adenoviral, retroviral) into the cell. Preferably,said vector is an expression vector and/or a gene transfer or targetingvector. Expression vectors derived from viruses such as retroviruses,vaccinia virus, adeno-associated virus, herpes viruses, or bovinepapilloma virus, may be used for delivery of the polynucleotides orvector of the invention into targeted cell populations. Preferably, saidcell is a germ line cell, embryonic cell, or egg cell or derivedtherefrom, most preferably said cell is a stem cell. As mentioned above,suitable gene delivery systems may include liposomes, receptor-mediateddelivery systems, naked DNA, and viral vectors such as herpes viruses,retroviruses, adenoviruses, and adeno-associated viruses, among others.Delivery of nucleic acids to a specific site in the body for genetherapy may also be accomplished using a biolistic delivery system, suchas that described by Williams (Proc. Natl. Acad. Sci. USA 88 (1991),2726-2729).

[0071] It is to be understood that the introduced polynucleotides andvectors express the gene product after introduction into said cell andpreferably remain in this status during the lifetime of said cell. Forexample, cell lines which stably express the polynucleotide under thecontrol of appropriate regulatory sequences may be engineered accordingto methods well known to those skilled in the art. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with the polynucleotide of the invention and aselectable marker, either on the same or separate plasmids. Followingthe introduction of foreign DNA, engineered cells may be allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows for the selection of cells havingstably integrated the plasmid into their chromosomes and grow to formfoci which in turn can be cloned and expanded into cell lines. Suchengineered cell lines are also particularly useful in screening methodsfor the detection of compounds involved in, e.g., T-cell activation orstimulation.

[0072] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, Cell11(1977), 223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska, Proc. Natl. Acad. Sci. USA 48 (1962), 2026), and adeninephosphoribosyltransferase (Lowy, Cell 22 (1980), 817) in tk⁻, hgprt⁻ oraprt⁻ cells, respectively. Also, antimetabolite resistance can be usedas the basis of selection for dhfr, which confers resistance tomethotrexate (Wigler, Proc. Natl. Acad. Sci. USA 77 (1980), 3567;O'Hare, Proc. Natl. Acad. Sci. USA 78 (1981), 1527), gpt, which confersresistance to mycophenolic acid (Mulligan, Proc. Natl. Acad. Sci. USA 78(1981), 2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, J. Mol. Biol. 150 (1981), 1); hygro, which confersresistance to hygromycin (Santerre, Gene 30 (1984), 147); or puromycin(pat, puromycin N-acetyl transferase). Additional selectable genes havebeen described, for example, trpB, which allows cells to utilize indolein place of tryptophan; hisD, which allows cells to utilize histinol inplace of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988),8047); and ODC (ornithine decarboxylase) which confers resistance to theornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO(McConlogue, 1987, In: Current Communications in Molecular Biology, ColdSpring Harbor Laboratory ed.).

[0073] In another embodiment the present invention relates to adiagnostic composition comprising any one of the above describedproteins, antibodies, (poly)peptides, polynucleotides, vectors or cellsand optionally suitable means for detection. The (poly)peptides andantibodies described above are, for example, suited for use inimmunoassays in which they can be utilized in liquid phase or bound to asolid phase carrier. Examples of immunoassays which can utilize said(poly)peptide are competitive and non-competitive immunoassays in eithera direct or indirect format. Examples of such immunoassays are theradioimmunoassay (RIA), the sandwich (immunometric assay) and theWestern blot assay. The (poly)peptides and antibodies can be bound tomany different carriers and used to isolate cells specifically bound tosaid polypeptides. Examples of well-known carriers include glass,polystyrene, polyvinyl chloride, polypropylene, polyethylene,polycarbonate, dextran, nylon, amyloses, natural and modifiedcelluloses, polyacrylamides, agaroses, and magnetite. The nature of thecarrier can be either soluble or insoluble for the purposes of theinvention.

[0074] There are many different labels and methods of labeling known tothose of ordinary skill in the art. Examples of the types of labelswhich can be used in the present invention include enzymes,radioisotopes, colloidal metals, fluorescent compounds, chemiluminescentcompounds, and bioluminescent compounds; see also the embodimentsdiscussed hereinabove.

[0075] Said diagnostic compositions may also be used for methods fordetecting expression of a polynucleotide of the invention by detectingthe presence of mRNA coding for a TIRC7 membrane protein which comprisesobtaining mRNA from a cell and contacting the mRNA so obtained with aprobe comprising a nucleic acid molecule of at least 15 nucleotidescapable of specifically hybridizing with a polynucleotide of theinvention under suitable hybridizing conditions (see also supra),detecting the presence of mRNA hybridized to the probe, and therebydetecting the expression of the TIRC7 protein by the cell.

[0076] Furthermore, the invention comprises methods of detecting thepresence of a TIRC7 membrane protein in a sample, for example, a cellsample, which comprises obtaining a cell sample from a subject,contacting said sample with one of the aforementioned antibodies underconditions permitting binding of the antibody to the TIRC7 protein, anddetecting the presence of the antibody so bound, for example, usingimmuno assay techniques such as radioimmunoassay or enzymeimmunoassay.Furthermore, one skilled in the art may specifically detect anddistinguish polypeptides which are functional TIRC7 proteins frommutated forms which have lost or altered their T-cell stimulatoryactivity by using an antibody which either specifically recognizes a(poly)peptide which has TIRC7 activity but does not recognize aninactive form thereof or which specifically recognizes an inactive formbut not the corresponding polypeptide having TIRC7 activity. Theantibodies of the present invention may also be used in affinitychromatography for purifying the TIRC7 membrane protein or abovedescribed (poly)peptides and isolating them-from various sources.

[0077] The invention also encompasses a method for diagnosing in asubject a predisposition to a disorder associated with the expression ofa TIRC7 allele which comprises isolating DNA from victims of the thedisorder associated with the under- or over-expression of a TIRC7protein; digesting the isolated DNA with at least one restrictionenzyme; electrophoretically separating the resulting DNA fragments on asizing gel; contacting the resulting gel with a nucleic acid probe asdescribed above capable of specifically hybridizing to DNA encoding aTIRC7 protein and labeled with a detectable marker; detecting labeledbands on the gel which have hybridized to the labeled probe to create aband pattern specific to the DNA of victims of the disorder associatedwith the expression of a TIRC7 protein; preparing the subject's DNAaccording to the above-mentioned steps to produce detectable labeledbands on a gel; and comparing the band pattern specific to the DNA ofvictims of the disorder associated with the expression of a TIRC7protein and the subject's DNA to determine whether the patterns are thesame or different and to diagnose thereby predisposition to the disorderif the patterns are the same. The detectable markers of the presentinvention may be labeled with commonly employed radioactive labels, suchas, for example, ³²P and ³⁵S, although other labels such as biotin ormercury as well as those described above may be employed as well.Various methods well-known to the person skilled in the art may be usedto label the detectable markers. For example, DNA sequences and RNAsequences may be labeled with ³²P or ³⁵S using the random primer method.Once a suitable detectable marker has been obtained, various methodswell-known to the person skilled in the art may be employed forcontacting the detectable marker with the sample of interest. Forexample, DNA-DNA, RNA-RNA and DNA-RNA hybridizations may be performedusing standard procedures. Various methods for the detection of nucleicacids are well-known in the art, e.g., Southern and northern blotting,PCR, primer extension and the like. Furthermore, the mRNA, cRNA, cDNA orgenomic DNA obtained from the subject may be sequenced to identifymutations which may be characteristic fingerprints of TIRC7 mutations indisorders associated with the expression of TIRC7 or mutated versionsthereof. The present invention further comprises methods, wherein such afingerprint may be generated by RFLPs of DNA or RNA obtained from thesubject, optionally the DNA or RNA may be amplified prior to analysis,the methods of which are well known in the art. RNA fingerprints may beperformed by, for example, digesting an RNA sample obtained from thesubject with a suitable RNA-Enzyme, for example RNase T₁, RNase T₂ orthe like or a ribozyme and, for example, electrophoretically separatingand detecting the RNA fragments on PAGE as described above or in theappended examples.

[0078] In a preferred embodiment, the pharmaceutical composition of thepresent invention comprises at least one second agent, preferably anagent which inhibits or activates T-cell stimulation depending on theintended use. Such agents include, for example, molecules that arecapable of blocking or mimicking receptor/ligand interaction or the likewhich leads to T-cell suppression and activation, respectively.

[0079] Such agents comprise those blocking the activity of, e.g.,costimulatory molecules, integrins, Ig-superfamily molecules, selectinsas well as drugs blocking chemokines and their respective receptorinteractions, drugs blocking IL2/IL2-receptor interaction and otherconventional immunosuppressive drugs such as TL-2R mAbs, IL-Toxins andIL-Muteins. Examples for costimulatory molecules and their ligands aredescribed in the prior art, e.g., in Schwartz, Cell 71 (1992),1065-1068. The interruption of the receptor/ligand interactions by usingmAbs or soluble CTLA4lg for the interaction between CD28 to the B7-2 andCTLA4 to B7-1 and B7-2 are described in Blazar, J. Immunol. 157 (1996),3250-3259; Bluestone, Immunity 2 (1995), 555-559; Linsley, Science 257(1992), 792-95. Examples for blocking the receptor/ligand interaction byusing mAbs to CD40 or CD40L are reported by Burden, Nature 381 (1996),434-435; Kirk, Proc. Natl. Acad. Sci. USA 94 (1997), 8789-8794. CD2antigen and its ligand LFA-3 are described in Bagogui Li et al., reviewin Adhesion Molecules, Fusion proteins, Novel Peptides, and MonoclonalAntibodies, Recent Developments in Transplantation Medicine, Vol. II,1995, Physicians&Scientists Publishing Co., Inc. and blocking of theirinteraction by using of mAbs (anti-Leu-5b, OKT11, T11) is reported inBrumberg, Transplantation 51 (1991) 219-225 or CD2.lgG1 fusion protein.The use of monoclonal Abs agains CD4 molecule is described in Cosimi,Surgery 108 (1990), 406-414. CD47 blockade by mAbs is described byRheinhold, J. Exp. Med. 185 (1997), 1-11. Integrins and lg-superfamilymolecules include LFA-1 with its ligand ICAM-1, -2, -3, Mac-1 with istligand ICAM-1, -3; ICAM-1 with its ligand LFA-1, Mac-1, CD43; ICAM-2with ist ligand LFA-1; ICAM-3 with its ligand LFA-1, Mac-1; VLA4 andVCAM-1 see, e.g., David, Adams, review in Adhesion Molecules, Fusionproteins, Novel Peptides, and Monoclonal Antibodies, Recent Developmentsin Transplantation Medicine, Vol. II, 1995, Physicians&ScientistsPublishing Co., Inc.; Isobe, Science, 255 (1992), 1125-1127; Cosimi, J.Immunology 144 (1990), 4604-4612; Hynes, Cell 69 (1992),11-25.

[0080] Furthermore selectively interfering agents with VLA-4 mAbs to thealpha4 integrin chain (CD49d) can be used, betal integrin chain (CD29),or an activation—induced neo-epitope of VLA-4 as well as soluble VLA-4ligands such as soluble fibronectin or its relevant peptide(GPEILDVPST), or soluble VCAM-1 or its relevant peptide. Moreselectively blocking agents are antisense oligonucleotides, designed toselectively hybridize with cytoplasmic alpha4, beta1, or VCAM-1 mRNA;Fedoseyeva, J. Immunol. 57 (1994), 606-612.

[0081] Another example is the drug pentoxifylline (PTX) that is able toblock expression of VCAM-1; Besler, J. Leukoc. Biol. 40 (1986), 747-754.Furthermore, VCAM-1 mAb, M/K-2, anti-murine, for example could prolongallograft survival, Orosz, Transplantation, 56 (1993), 453-460.

[0082] Blocking of members of the integrin family and their ligands byusing mAbs is decribed in Kupiec-Weglinski, review in AdhesionMolecules, Fusion proteins, Novel Peptides, and Monoclonal Antibodies,Recent Developments in Transplantation Medicine, Vol. II, 1995,Physicians&Scientists Publishing Co., Inc.

[0083] Selectins, e.g., L-selectin (CD62L), E-selectin (CD62E),P-selectin (CD62P) have been described in Forrest and Paulson, Selectinfamily of adhesion molecules. In: Granger and Schmid-Schonbein, eds.Physiology and Pathophysiology of Leukocyte Adhesion. New York, OxfordPress, 1995, pp 68-146.

[0084] The combination of conventional immunosuppressive drugs, e.g.,ATG, ALG, OKT3, Azathioprine, Mycophenylate, Mofetyl, Cyclosporin A,FK506, Corticosteroids may be used as decribed in Cosimi,Transplantation 32 (1981), 535-539; Shield, Transplantation 38 (1984),695-701.

[0085] The interruption of chemokines and interactions with theirrespective receptor by using mAbs is reviewed in Luster,Chemokines-chemotactic cytokines that mediate inflammation, New Engl. J.Med. Feb. (1998), 436-445.

[0086] Thus, any agent as defined above and referenced by way of examplecan be used in accordance with the pharmaceutical composition of theinvention or the methods and uses described hereinbelow. On the otherhand, it is evident to the person skilled in the art that thepolynucleotides, vectors, proteins, peptides, polypeptides, antibodies,cells, and pharmaceutical compositions of the invention can be used formethods and uses described for the above referenced T-cell costimulatorymolecules, inhibitors and drugs.

[0087] Advantageously, the pharmaceutical composition of the inventionis intended for use in organ transplantation, for the treatment ofautoimmune, allergic or infectious diseases, or for the treatment oftumors. An example for the use of the pharmaceutical composition of theinvention for improving allograft or xenograft tolerance is describedwith respect to administration of an LFA-3 and CD2 binding protein,respectively, in WO93/06852.

[0088] Furthermore, this invention pertains to methods for modulating(antigen-specific) T-cell unresponsiveness. The term “T-cellunresponsiveness” as used herein refers to a reduction in or lack ofT-cell proliferation, lymphokine secretion or induction of effectorfunctions by a T-cell upon exposure to the antigen (or antigenicportion). The pharmaceutical compositions of this invention provide ameans for inducing, maintaining or reversing unresponsiveness of aT-cell to an antigen in vitro or in vivo. Accordingly, the compounds ofthis invention are particularly useful for modulating (antigen-specific)T-cell unresponsiveness. The term “modulation” is intended to includeboth inducing and maintenance of an unresponsive state and reversal ofan unresponsive state, i.e., restoration of T-cell responsiveness.

[0089] As is described in Example 3, after anti-TIRC7 antibody treatmentT-cells remain in an unresponsive, but functional state since exogenousrecombinant IL-2 reversed the antiproliferative effect of theantibodies. Thus, in another embodiment the present invention relates toan in vitro method for inducing or maintaining unresponsiveness of aT-cell to an antigen comprising contacting the T-cell with an agentwhich inhibits stimulation of the T-cell through a TIRC7 membraneprotein. Recipes for how to carry out methods for modulation T-cellunresponsiveness are generally known to the skilled person and aredescribed in, e.g., WO95/24217 and references cited therein.

[0090] Hence, the present invention also relates to the use of an agentwhich inhibits T-cell stimulation through a TIRC7 membrane protein forthe preparation of a pharmaceutical composition for inducing ormaintaining T-cell unresponsiveness to an antigen in a subject. Themethods and uses of the invention may be used with primed or unprimedT-cells depending on what is intended by the person skilled in the art.

[0091] Preferably, in the method or the use of the invention the agentblocks an interaction of the TIRC7 membrane protein with its ligand. Asis described above, the results of the experiments performed within thescope of the present invention suggest the existence of a ligandinteracting with the TIRC7 protein and thereby stimulating T-cellproliferation. Blocking said interaction, e.g. with antibodies orsoluble (poly)peptides derived from the TIRC7 membrane protein shouldresult in T cell unresponsiveness. Thus, in a preferred embodiment ofthe method or the use of the invention the agent is a polynucleotide, avector, a cell, peptide or polypeptide, or antibody describedhereinbefore.

[0092] In another preferred embodiment of the invention, the abovedescribed method or use further comprise the use of a second agent asdefined above.

[0093] In another embodiment, the present invention relates to apharmaceutical composition comprising a first agent which stimulates aT-cell through a TIRC7 membrane protein, and optionally apharmaceutically acceptable carrier. As is immediately evident to theperson skilled in the art, the provision of the novel TIRC7 protein ofthe invention opens up the way of alternative approaches for T-cellstimulation and treating corresponding diseases. The agent thatstimulates the T-cell through the TIRC7 membrane protein is expected tomarkedly enhance the proliferation of (activated) T-cells and thus iscapable of augmenting the immune response. Examples for this type of“Vaccine” is described, e.g., in WO91/11194 and in the literature, e.g.,referred to above. Such agents also comprise promoters which can beinserted in front of the coding region of the TIRC7 protein encodinggene, e.g., via gene transfer and homologous recombination in the 5′untranslated region of the gene, see also supra. Such promoter may beregulated and thus permit the controlled expression of the TIRC7 proteinin certain cells.

[0094] Preferably, said agent is a ligand of the TIRC7 membrane proteinor is at least one anti-TIRC7 membrane protein antibody described above.

[0095] In a preferred embodiment of the invention, the pharmaceuticalcomposition further comprises a second agent which stimulates T-cellproliferation, for example IL-2, IL-4 or an agent which stimulates aT-cell through a CD2, CD28, CD40 or CTLA4 surface receptor.

[0096] In a further embodiment the present invention relates to an invitro method for restoring responsiveness to an antigen by a T-cellwhich is unresponsive to the antigen, comprising contacting the T-cellin the presence of the antigen with a first agent which stimulates theT-cell through a TIRC7 membrane protein. Besides the application of theligand itself said TIRC7 ligand may be preferably expressed on the cellsurface by introducing into the cell a nucleic acid molecule encodingthe TIRC7 ligand in a suitable form for expression of the TIRC7 ligandon the cell surface. Preferably the cell is a tumor cell. Vectors andmethods for the introduction of such, nucleic acid molecules are wellknown to the skilled person and are also described, e.g., above.

[0097] The method of the invention can further comprise contacting theT-cell with a second agent as defined for the pharmaceuticalcompositions above. Preferably, the T-cell is contacted with the secondagent prior to being contacted with the first agent.

[0098] In a preferred embodiment of the method of the invention, thesecond agent, preferably CD2, CD28, CTLA4 ligand or CD40 is expressed onthe cell surface by introducing into the cell a nucleic acid moleculeencoding the CD2, CD28, CTLA4 ligand or CD40 in a form suitable forexpression of said ligand on the cell surface.

[0099] In another embodiment, the present invention relates to the useof a first agent as defined above, which is capable of stimulating aT-cell through a TIRC7 membrane protein for the preparation of apharmaceutical composition for stimulating a T-cell response to a tumorcell in a subject with a tumor. Such agents comprise, for example, alsobispecific antibodies described supra. Preferably the tumor cell ismodified to express a TIRC7 ligand and/or a CD2, CD28, CTLA4 ligand orCD40 preferably on the tumor cell surface.

[0100] In a preferred embodiment of the use the invention the tumor cellis obtained from the subject, modified ex vivo to form a modified tumorcell and said modified tumor cell is used for the preparation of apharmaceutical composition which is designed for administration to thesubject.

[0101] In another preferred embodiment of the use of the invention theT-cells are obtained from a subject, contacted with IL-2 or IL-4 ex vivoand said modified T-cells are used for the preparation of pharmaceuticalcomposition which is designed for the administration to the subject.

[0102] In a particularly preferred embodiment of the invention, theuses, methods and pharmaceutical compositions are intended to be appliedto a subject who is a recipient of bone marrow transplant or peripheralstem cell transplant. Preferably the pharmaceutical composition isdesigned for contacting with bone marrow or peripheral stem cell priorto transplantation into the recipient.

[0103] In a further particular preferred embodiment, the methods anduses of the present invention are applied in organ grafttransplantation, peripheral stem cell transplantation or for thetreatment of auto-immune diseases or allergy.

[0104] Furthermore, the invention relates to a method for identifyingT-cell activating or co-stimulating compounds or for identifyinginhibitors of T-cell activation and stimulation comprising

[0105] (a) culturing T-cells in the presence of the TIRC7 protein,(poly)peptide, antibody, or cell described above and, optionally, in thepresence of a component capable of providing a detectable signal inresponse to T-cell activation, with a compound to be screened underconditions permitting interaction of the compound with the TIRC7protein, (poly)peptide, antibody or cell(s); and

[0106] (b) detecting the presence or absence of a signal generated fromthe interaction of the compound with the cells.

[0107] The term “compound” in the method of the invention includes asingle substance or a plurality of substances which may or may not beidentical.

[0108] Said compound(s) may be comprised in, for example, samples, e.g.,cell extracts from, e.g., plants, animals or microorganisms.Furthermore, said compounds may be known in the art but hitherto notknown to be capable of inhibiting T-cell activation or not known to beuseful as a T-cell costimulatory factor, respectively. The plurality ofcompounds may be, e.g., added to the culture medium or injected into thecell.

[0109] If a sample containing (a) compound(s) is identified in themethod of the invention, then it is either possible to isolate thecompound from the original sample identified as containing the compound,in question or one can further subdivide the original sample, forexample, if it consists of a plurality of different compounds, so as toreduce the number of different substances per sample and repeat themethod with the subdivisions of the original sample. It can then bedetermined whether said sample or compound displays the desiredproperties by methods known in the art such as described herein and inthe appended examples. Depending on the complexity of the samples, thesteps described above can be performed several times, preferably untilthe sample identified according to the method of the invention onlycomprises a limited number of or only one substance(s). Preferably saidsample comprises substances of similar chemical and/or physicalproperties, and most preferably said substances are identical. Themethods of the present invention can be easily performed and designed bythe person skilled in the art, for example in accordance with other cellbased assays described in the prior art (see, e.g., EP-A-0 403 506) orby using and modifying the methods as described in the appendedexamples. Furthermore, the person skilled in the art will readilyrecognize which further compounds and/or cells may be used in order toperform the methods of the invention, for example, B-cells,interleukins, or enzymes, if necessary, that, e.g., convert a certaincompound into the precursor which in turn stimulates or suppressesT-cell activation or that provide for (co)stimulatory signals. Suchadaptation of the method of the invention is well within the skill ofthe person skilled in the art and can be performed without undueexperimentation.

[0110] Compounds which can be used in accordance with the method of thepresent invention include peptides, proteins, nucleic acids includingcDNA expression libraries, antibodies, small organic compounds, ligands,peptidomimetics, PNAs and the like. Said compounds can also befunctional derivatives or analogues of known T-cell activators orinhibitors. Methods for the preparation of chemical derivatives andanalogues are well known to those skilled in the art and are describedin, for example, Beilstein, Handbook of Organic Chemistry, Springeredition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. andOrganic Synthesis, Wiley, N.Y., USA. Furthermore, said derivatives andanalogues can be tested for their effects according to methods known inthe art or as described, for example, in the appended examples.Furthermore, peptidomimetics and/or computer aided design of appropriateactivators or inhibitors of T-cell activation can be used, for example,according to the methods described below. Appropriate computer programscan be used for the identification of interactive sites of a putativeinhibitor and the TIRC7 protein by computer assistant searches forcomplementary structural motifs (Fassina, Immunomethods 5 (1994),114-120). Further appropriate computer systems for the computer aideddesign of protein and peptides are described in the prior art, forexample, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak,Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986),5987-5991. The results obtained from the above-described computeranalysis can be used in combination with the method of the inventionfor, e.g., optimizing known T-cell activators or inhibitors. Appropriatepeptidomimetics can also be identified by the synthesis ofpeptidomimetic combinatorial libraries through successive chemicalmodification and testing the resulting compounds, e.g., according to themethods described herein and in the appended examples. Methods for thegeneration and use of peptidomimetic combinatorial libraries aredescribed in the prior art, for example in Ostresh, Methods inEnzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996),709-715. Furthermore, the three-dimensional and/or crystallographicstructure of inhibitors or activators of T-cell stimulation can be usedfor the design of peptidomimetic inhibitors or activators of T-cellactivation to be tested in the method of the invention (Rose,Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4(1996), 1545-1558).

[0111] In summary, the present invention provides methods foridentifying compounds which are capable of modulating T-cell mediatedimmune responses. Accordingly compounds identified in accordance withthe method of the present invention to be inhibitors and activators,respectively, of T-cell stimulation or activation are also within thescope of the present invention.

[0112] Compounds found to activate T-cell mediated responses may be usedin the treatment of cancer and related diseases. In addition, it mayalso be possible to specifically inhibit viral diseases, therebypreventing viral infection or viral spread. Compounds identified assuppressors of T-cell activation or stimulation can be used, e.g., fortreating skin conditions (see, e.g., WO93/06866) or in allogenic orxenogenic cell or organ transplantation in order to avoid graftrejection; see also supra.

[0113] The compounds identified or obtained according to the method ofthe present invention are thus expected to be very useful in diagnosticand in particular for therapeutic applications. Hence, in a furtherembodiment the invention relates to a method for the production of apharmaceutical composition comprising formulating and optionallysynthesizing the compound identified in step (b) of the above describedmethod of the invention in a pharmaceutically acceptable form. Hence,the present invention generally relates to a method of making atherapeutic agent comprising synthesizing the proteins, (poly)peptides,polynucleotides, vectors, antibodies or compounds according to theinvention in an amount sufficient to provide said agent in atherapeutically effective amount to the patient. Methods forsynthesizing these agents are well known in the art and are described,e.g. above.

[0114] The therapeutically useful compounds identified according to themethod of the invention may be administered to a patient by anyappropriate method for the particular compound, e.g., orally,intravenously, parenterally, transdermally, transmucosally, or bysurgery or implantation (e.g., with the compound being in the form of asolid or semi-solid biologically compatible and resorbable matrix) at ornear the site where the effect of the compound is desired. Therapeuticdoses are determined to be appropriate by one skilled in the art, seealso supra.

[0115] Such useful compounds can be for example transacting factorswhich bind to the TIRC7 protein of the invention. Identification oftransacting factors can be carried out using standard methods in the art(see, e.g., Sambrook, supra, and Ausubel, supra). To determine whether aprotein binds to the TIRC7 protein of the invention, standard nativegel-shift analyses can be carried out. In order to identify atransacting factor which binds to the TIRC7 of the invention, thepolypeptides and peptides of the invention can be used as an affinityreagent in standard protein purification methods, or as a probe forscreening an expression library. Once the transacting factor isidentified, modulation of its binding to the TIRC7 protein of theinvention can be pursued, beginning with, for example, screening forinhibitors against the binding of the transacting factor to the TIRC7protein of the present invention. Activation or repression of TIRC7specific genes could then be achieved in subjects by applying thetransacting factor (or its inhibitor) or the gene encoding it, e.g., ina vector described in the embodiments hereinbefore. In addition, if theactive form of the transacting factor is a dimer, dominant-negativemutants of the transacting factor could be made in order to inhibit itsactivity. Furthermore, upon identification of the transacting factor,further components in the pathway leading to activation (e.g. signaltransduction) or repression of a gene encoding the TIRC7 protein of thepresent invention can then be identified. Modulation of the activitiesof these components can then be pursued, in order to develop additionaldrugs and methods for modulating the expression or activity of the TIRC7protein of the present invention.

[0116] Beside the above described possibilities to use thepolynucleotides according to the invention for gene therapy and theiruse to identify homologous molecules, the described polynucleotides mayalso be used for several other applications, for example, for theidentification of nucleic acid molecules which encode proteins whichinteract with the TIRC7 protein described above. This can be achieved byassays well known in the art, for example, as described in Scofield(Science 274 (1996), 2063-2065) by use of the so-called yeast“two-hybrid system”. In this system the (poly)peptide encoded by thepolynucleotides according to the invention or a smaller part thereof islinked to the DNA-binding domain of the GAL4 transcription factor. Ayeast strain expressing this fusion protein and comprising a lacZreporter gene driven by an appropriate promoter, which is recognized bythe GAL4 transcription factor, is transformed with a library of cDNAswhich will express animal, preferably mammal proteins or peptidesthereof fused to an activation domain. Thus, if a peptide encoded by oneof the cDNAs is able to interact with the fusion protein comprising a(poly)peptide of the invention, the complex is able to direct expressionof the reporter gene. In this way the polynucleotide according to theinvention and the encoded peptide can be used to identify peptides andproteins interacting with TIRC7 proteins.

[0117] Other methods for identifying compounds which interact with theTIRC7 protein according to the invention or nucleic acid moleculesencoding such molecules are, for example, the in vitro screening withthe phage display system as well as filter binding assays or “real time”measuring of interaction using, for example, the BIAcore apparatus(Pharmacia); see references cited supra.

[0118] Furthermore, the present invention relates to the use of thepolynucleotide, the vectors, peptides, polypeptides, antibodies andcells of the invention as well as compounds identified in accordancewith a method of the invention described hereinabove for the preparationof a pharmaceutical composition for the treatment of diseases involvingT-cell activation and associated with Th1 and Th2 immune response, forthe treatment of acute and chronic rejection of allo- and xeno organtransplants and bone marrow transplantation, for the treatment ofrheumatoid arthritis, lupus erythramatodes, multiple sklerosis,encephalitis, vasculitis, diabetes mellitus, pancreatitis, gastritis,thyroiditis, for the treatment of maligne disorders of T, B or NK cells,for the treatment of asthma, lepramatosis, Helicobacter pyloriassociated gastritis or for the treatment of skin tumors, adrenal tumorsor lung tumors.

[0119] The polynucleotides, vectors, cells, proteins, (poly)peptides,antibodies, inhibitors, activators, pharmaceutical compositions, usesand methods of the invention can be used for the treatment of all kindsof diseases hitherto unknown as being related to or dependent on themodulation of T-cells. The pharmaceutical compositions, methods and usesof the present invention may be desirably employed in humans, althoughanimal treatment is also encompassed by the methods and uses describedherein.

[0120] These and other embodiments are disclosed and encompassed by thedescription and Examples of the present invention. Further literatureconcerning any one of the antibodies, methods, uses and compounds to beemployed in accordance with the present invention may be retrieved frompublic libraries and databases, using for example electronic devices.For example the public database “Medline” may be utilized which isavailable on the Internet, for example underhttp://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases andaddresses, such as http://www.ncbi.nlm.nih.gov/,http://www.infobiogen.fr/,http://www.fmi.ch/biology/research_tools.html, http://www.tigr.org/, areknown to the person skilled in the art and can also be obtained using,e.g., http://www.lycos.com. An overview of patent information inbiotechnology and a survey of relevant sources of patent informationuseful for retrospective searching and for current awareness is given inBerks, TIBTECH 12 (1994), 352-364.

BRIEF DESCRIPTION OF DRAWINGS

[0121] This disclosure may best be understood in conjunction with theaccompanying drawings, incorporated herein by references, which show:

[0122]FIG. 1: Identification of TIRC7 from alloactivated T cells. (A).Differential display identification of a 350 bp transcript upregulated24 hours after alloactivation of human T lymphocytes. Each lane shows amRNA expression pattern from a one-way MLR at either 0 or 24 h afteractivation. Two different MLRs (a and b) exhibited similar patterns ofgene expression. (B). Nucleotide sequence of TIRC7 cDNA. The cDNA anddeduced 614 amino acid sequences of the TIRC7 transcript is shown.Predicted transmembrane regions are underlined and bold.

[0123]FIG. 2: Predicted secondary structure of TIRC7. The predictedsecondary structure of TIRC7 protein contains seven transmembranespanning domains (TM). Peptides (P1-P7) synthesized according tosequences in the putative intracellular amino terminus (NH₃),extracellular carboxy terminus (COOH), and the largest intracellular(IC) and extracellular (EC) loop were used to raise rabbit anti-TIRC7polyclonal antibodies. Anti-TIRC7 antibodies with T cell responsemodulatory effect are given in the box.

[0124]FIG. 3: TIRC7 is upregulated in T cell activation and is localizedon the cell membrane. (A). TIRC7 mRNA expression is upregulated inallostimulated human T cells. MLR-0 and MLR-24 indicate time points 0 hand 24 h, respectively, after coincubation of allogenic responder andstimulator lymphocytes. (B). TIRC7 mRNA upregulation in MLR (24h) isprevented by Cyclosporin A (Cyc A).

[0125]FIG. 4: Localization of TIRC7 protein. (A). A single 75 kDaprotein is detected by anti-TIRC7 antibodies (Ab79 is shown)predominantly in membrane protein extracts of human lymphocytes. Bindingof Ab79 to TIRC7 is abolished in the presence of the respective peptideP2 (Ab+P2). (B). The same single 75 kDa protein is also detected by ananti-c-miyc antibody in membrane preparations of COS-7 cells transientlytransfected with a c-myc tagged TIRC7 expression vector (COS7-t) but notin untransfected COS-7 cells (COS7-nt).

[0126]FIG. 5: Anti-TIRC7 antibodies inhibit T cell proliferation andIL-2 production. (A). Anti-TIRC7 antibodies (Ab73, Ab76, Ab79) directedagainst extracellularly located TIRC7 peptides (P4, P6, P2) inhibitproliferation in alloantigen stimulated T cells as determined by[³H]-thymidine incorporation. Inhibition was diminished when antibodieswere preincubated with their respective peptides. Proliferation in a MLRafter 24 h is displayed as positive control. Proliferation was notaffected by preimmune serum (control Ab). Each bar represents mean andSD from seven independent experiments. (B). Inhibition of proliferationby exogenous TIRC7 protein. In a one-way MLR in vitro translated TIRC7protein inhibited proliferation in a dose-dependent manner. Proteindilutions are indicated. No inhibition of proliferation was observedwhen vector alone (vector) or an unrelated cDNA (unrel) was used in thein vitro translation preparation. (C). Anti-TIRC7 antibodies inhibit Th1specific cytokine expression. PHA stimulated human lymphocytes werecoincubated with Ab73, Ab76 and Ab79, respectively. Supernatants ofmitogen stimulated cultures were taken at 24 and 48 h. Cytokineexpression in the supernatants were determined by ELISA. Each barrepresents mean and SD from three independent experiments. (D).Anti-TIRC7 antibody mediated inhibition of proliferation of PHAactivated T cells is reconstituted by exogeneous IL-2. Each barrepresents mean and SD from three independent experiments.

[0127]FIG. 6: Anti-TIRC7 antibody targeting in vivo significantlyprolongs allograft survival. (A). Lewis rat recipients of Wistar Furthrat kidney allografts received either anti-TIRC7 Ab73 (n=7), controlantibody from preimmune serum (n=7), or no treatment (n=7). Treatmentwas initiated at 2 h prior to and immediately after transplantation, andwas repeated on day 1, 2, 4, and 6 post-transplantation. Animals treatedwith control antibody showed a mean survival time of 8±1 days whereasmean survival time of animals representing the untreated control groupwas 7±2 days. Six of the seven animals in the experimental groupmaintained functional grafts for more than 45 days. One rat in theanti-TIRC7 antibody treated experimental group had a survival time of 21days. As assessed at day 45 after transplantation, the mean survivaltime in this group was 41,5 days (p<0.001 vs controls).

[0128]FIG. 7: Histological analysis of kidney allografts at day 7post-transplantation. (A). Kidney allografts of rats receiving controlantibodies showed severe tissue destruction and diffuse mononuclearinfiltration which was similar to histological findings in the kidneyallografts of untreated animals. (B). Renal allografts of two additionalanti-TIRC7 antibody treated animals sacrificed at day 7 showed very mildinterstitial infiltration of mononuclear cells. Tissue lessions were notidentified in the allografts of these animals.

[0129] A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

EXAMPLES Example 1

[0130] Cloning of a Novel Membrane Protein (TIRC7) Encoding a Gene thatIs Differentially Expressed in Alloactivated Human T Cells

[0131] To identify novel genes induced during the early stages of T cellactivation in response to alloantigens, differential display RT-PCRanalysis of mRNA expression was performed at time 0 and 24 h afterinitiation of a human mixed lymphocyte culture (MLR). In conformancewith institutional policies regarding human experimentation, peripheralblood lymphocytes (PBLs) were isolated from healthy human volunteersusing standard Ficoll centrifugation methods and diluted into RPMIcontaining 10% fetal calf serum. Responder PBLs were stimulated withequal numbers of irradiated (3000 rad, 13 min) stimulator PBLs. Cellswere co-cultured for 24 h in tissue flasks at an initial concentrationof 10⁶ cells/ml for RNA isolation. Total RNA was isolated from MLR at 0and 24 h using the RNAzol B method (Tel-Test, Inc) and differentialdisplay was performed as described previously (Kojima et al., 1996).Briefly, 2 mg of total RNA was reverse transcribed using an oligo-dTprimer and 200 U MMLV reverse transcriptase (Gibco/BRL). A 40 cycle PCRamplification with a total volume of 10 ml was performed by using 1 mgof cDNA, 1.25 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 nMprimer, 5 mCi ³⁵S-dATP, and 0.3 U Taq polymerase. The primers for thePCR amplification were: 5′-GACGGAACAGCTTC-3′ (SEQ ID No. 10) and5′-TGCGTCTGGTTCT-3′ (SEQ ID No. 11). The PCR products were stored at 4°C. and separated by electrophoresis in 6% polyacrylamide-urea gels,transferred to filter paper, dried, and autoradiographed. Thedifferentially expressed cDNA fragment was excised from the gel, eluted,reamplifed, cloned into pBluescriptSK⁺ plasmid, and sequenced at theHoward Hughes Biopolymers Research Facility or the Dana Farber CancerInstitute Biopolymer Facility at Harvard Medical School. Homologysearches were performed using BLAST at NCBI. Alignments were performedusing Geneworks 2.1.1. As shown in FIG. 1A, a 350 bp cDNA fragment wasidentified which was induced at 24 h after stimulation, and was used toidentify several clones from l-gt-10 cDNA libraries from human T cells.A l gt-10 cDNA library (Clontech) prepared from human T cells activatedfor 48 hr with PHA was screened with the 350 bp TIRC7 cDNA fragment.Briefly, plaque lifts of 1,200,000 independent cDNA clones werehybridized with a ³²P-labeled cDNA for 24 hrs at 42° C. in 40%formamide, 10% dextran sulfate, 4×SSC (1×SSC consists of 150 mM NaCl, 15mM sodium citrate, pH 7.0), 0.8×Denhardt's solution (1×Denhardt'scontains of 0.02% polyvinylpyrolidone, 0.02% Ficoll, 0.02% bovine serumalbumin), 0.5% sodium dodecyl sulfate (SDS), and 20 mg salmon sperm DNA.The filters were washed twice for 20 min at room temperature with 2×SSC,10% SDS and for 30 min at 65° C. with 0.2×SSC, 10% SDS followed byautoradiography. Three positive clones were selected and plaquepurified. cDNA was sequenced in both directions using a primer walkingstrategy. A PAC genomic library was screened using a 2 kb cDNA probecontaining the ORF cDNA of TIRC7, and the entire genomic cDNA of TIRC7and OC116 was bidirectinally sequenced. Sequence analysis revealed a2488 bp cDNA (SEQ ID NO: 1) which was designated as TIRC7 (T cell immuneresponse cDNA7; Gen Bank Accession Number: AF025374), containing an openreading frame of 1842 nt and predicting a protein length of 614 aminoacids (SEQ ID No. 2) (FIG. 1B). Furthermore, a second cDNA of TIRC7 wascloned (SEQ ID No. 12) encoding an amino acid sequence that is identicalto that of the protein encoded by the other TIRC7 cDNA except for oneamino acid substitution at amino acid position 121 (Arg® Gln). It wastherefore concluded that both cDNAs represent alleles of the TIRC7encoding gene. Hydrophobicity analysis of the protein sequence revealedseven hydrophobic domains, compatible with transmembrane spanningdomains. The N-terminus of TIRC7 lacks a consensus signal peptidesequence and followed by seven hydrophobic domains predicting a topologyof an intracellular N-terminus and extracellularly oriented C-domain(FIG. 2). TIRC7 contains multiple putative sites of post-translationalmodification including phosphorylation sites for PKC (at amino acids 58,98, and 148) and PKA (at amino acid 21), as well as N-linkedglycosylation sites (at amino acid 267 and 287). No amino acid homologywas found with any proteins known to be involved in T cell activation.TIRC7 does share amino acid homology (12-83%) with several proteinsreported as putative subunits of the vacuolar proton pump H⁺-ATPase(VPP) in a variety of species (Bowman et al., 1988; Lee et al., 1990;Peng et al., 1994; Perin et al., 1991, Manolson et al., 1992, Manolsonet al., 1994; Solioz and Davies., 1994; Li et al., 1996). Analysis ofthe complete genomic DNA organization of TIRC7 revealed that TIRC7 and arecently reported human osteoclast specific cDNA, named OC116 (Li etal., 1996), are alternatively spliced transcripts of the same gene. Thefunction of OC116 is so far unknown and the 2640 nt mRNA, encoding a 814residue protein, was demonstrated to be exclusively expressed in humanosteoclast cells. The regions of strongest homology between TIRC7 andthese putative VPPs are predominantly in their predicted transmembranedomains and the C-termini. Thus, TIRC7 belongs to a larger family ofstructurally related membrane proteins whose functions have not beenclearly elucidated.

[0132] To determine the expression kinetics of TIRC7, Northern blotanalysis of total RNA from alloantigen activated lymphocytes wasperformed. Northern blots were prepared with 7-10 mg of total RNA asdescribed previously (Kojima et al., 1996). Poly(A)⁺ Northern blotscontaining RNA from various human tissues were purchased from Clontech.Northern blots were probed with the full-length TIRC7 cDNA or aTIRC7-specific cDNA fragment (nt 52-391). Overnight hybridizations wereperformed with ³²P labeled cDNA probes (10⁶ cpm/ml) at 42° C. in 40%formamide, 10% dextran sulfate, 4×SSC, 7 mM Tris (pH 7.6),0.8×Denhardt's solution, 0.02 mg/ml salmon sperm DNA, and 10% SDS. Blotswere washed twice in 2×SSC and 0.1% SDS for 20 min at room temperature,once at 65° C. in 0.2×SSC, 0.1% SDS and autoradiographed at −80° C. ATIRC7 specific cDNA probe detected the expected 2.5 kb transcript aswell as an additional 4 kb mRNA of unknown origin (FIGS. 3A-B).Alloactivation of T cells resulted in a 20-fold upregulation of TIRC7expression at 24 h (FIG. 3A). TIRC7 expression was transient with noincrease at 1 h, peak expression at 24 h, and a return to baseline at 72h. To demonstrate that the upregulation of TIRC7 occurred in theresponder T cell population, an additional MLR was performed usingstimulators depleted of T cells. For studies on induction or inhibitionof TIRC7 expression, PBLs were exposed to concanavalin A (10 ng/ml),phytohemagglutinin (PHA) (20 mg/ml), Staphylococcus aureus enterotoxin B(10 mg/ml), OKT3-mAb (10 mg/ml), cyclosporine A (1 mg/ml) or rIL-2 (10U/ml). The stimulation with OKT3-mAb was carried out by immobilizing theantibody on plastic culture plates overnight at 4° C. before adding thecell suspension. For RNA isolation from CD4⁺ and CD8⁺ human T cells PBLswere incubated with immunomagnetic beads coated with anti-CD4 oranti-CD8 TgG and then subjected to magnetic separation. TIRC7 mRNAexpression was found to be increased in responder T cells 24 h afterco-culture.

[0133] Cyclosporine A (Cyc A), an inhibitor of the calcineurin dependentT cell activation pathways, blocked the induction of TIRC7 in a MLR(FIG. 3B). Furthermore, exogenous IL-2 was a potent inducer of TIRC7expression, whereas a modest increase in TIRC7 expression was observedwith Staphylococcus aureus enterotoxin B (SEB) or OKT3-mAb stimulationafter 24 h, though OKT3-mAb increased TIRC7 expression after 48 h tosimilar levels induced by alloantigen. In contrast, neither concanavalinA (ConA) nor phytohemagglutinin (PHA) increased TIRC7 expression at 24or 48 h.

[0134] Northern analysis revealed that TIRC7 is almost exclusivelyexpressed in immune tissues and exhibits high levels of mRNA expressionin spleen, lymph nodes, peripheral blood, and appendix, whereas lowerlevels of expression are observed in bone marrow, fetal liver andthymus, respectively. TIRC7 was also detected in CD4⁺ and CD8⁺lymphocytes but not in EBV-transformed primary B cells, Burkitt'slymphoma cells, EBV-infected Burkitt's lymphoma cells, and resting oractivated Jurkat cells.

[0135] In Western blot analysis, a single protein of approximately 75kDa molecular mass was detected predominantly in membrane extracts ofhuman lymphocytes (FIG. 4A). PBLs were lysed in 50 mM Tris (pH 7.59),150 mM NaCl, 1% triton, aprotinin (0.15 U/ml), 20 mM leupeptin, and 1 mMphenlymethylsulfonylfluoride. Lysates were separated by SDS-PAGE andtransferred to a nitrocellulose membrane. The membranes were incubatedwith primary (see Example 2, infra) and secondary antibody for 1 h each,and bound primary antibody was detected by horseradishperoxidase-conjugated secondary antibody followed by enhancedchemiluminescence (Boehringer). Polyclonal Ab79 (see Example 2, infra)was used at 1:5000 dilution. The secondary antibody (anti-rabbit-IgG)conjugated with peroxidase was used at 1:2000 dilution. To detect theTIRC7/c-miyc fusion protein the anti-c-myc antibody was used at 1:1000dilution and the secondary antibody (anti-mouse-IgG) used at 1:2000dilution. The same protein was found in membrane preparations from CHOcells and COS-7 cells (FIG. 4B) stably and transiently transfected witha c-myc tagged TIRC7 expression vector, respectively. The full-lengthTIRC7 ORF was cloned upstream of a c-myc epitope sequence to create afusion protein construct in a mammalian expression vector (Progmega).Transient transfection of COS7 cells and stable transfection of CHOcells was performed by lipofectamine transfection method as described(Schülein et al., 1996). An anti-c-myc antibody (InVitrogen) was used todetect the protein. TIRC7 localization to the cell membrane wasconfirmed by confocal microscopy and flow cytometric analysis in humanlymphocytes as well as in stably transfected CHO cells.

Example 2

[0136] TIRC7 Mediates an Essential Signal During Early Events of T CellActivation

[0137] The functional significance of a number of proteins required in Tcell activation has been determined by modulation of their signaling bytargeting with specific antibodies. To examine whether antibodiesdirected against TIRC7 could alter the T cell proliferative response,seven synthetic peptides representing different domains of the TIRC7protein (P1-P7, 12 mers; SEQ ID Nos. 3 to 9) were used to producepolyclonal rabbit antipeptide antibodies (FIG. 2A). Antigenicnon-transmembrane regions of TIRC7 were identified using PSORT andPC-GENE and used to design short peptide sequences. Purified syntheticpeptides (P1-P7) (Laboratories of Henklein, Berlin, Germany) were usedfor immunization of rabbits (Seramun, Berlin, Germany). Animals wereboostered after three and six weeks. A total of 14 polyclonal antibodieswere prepared against 7 different peptides (P1-P7). The pooled antiserawere purified by affinity chromatography after binding of peptide toBSA. All antibodies were tested by ELISA with their respective peptides.As shown in FIG. 5A, three of the antibodies Ab73, Ab76 and Ab79, whichwere directed against the extracellularly located domains P4, P6, and P2(SEQ ID Nos. 6, 8 and 2), respectively, were found to inhibit theproliferation of alloactivated T cells by 87-93%. For proliferationassays, responder PBLs were plated in the presence of an equal number ofirradiated-stimulator cells (total of 2×10⁶ cells/ml) with either mediaalone, antibodies or control serum into each well of a round-bottomed96-well microtiter plate in a final culture volume of 200 ml. Anti-TIRC7anti-sera were added in 1:500 dilutions to MLR. The plates wereincubated at 37° C., 5% CO₂ and pulsed for the final 18 h of the culturewith 1 mCi [³H]-thymidine (ICN Biochemicals) per well. All plates wereharvested and counted on a Betaplate liquid scintillation counter.Counts were represented as the mean cpm of quadruplicate wells harvestedat 72 h following the 18 hr pulse. To prevent complement lysis of thecells, antibodies were incubated at 52° C. for 20 min prior to use.Peptide blocking experiments were performed by coincubation of theantibody with the appropriate peptide for 30 min at 4° C. prior toinitiating the MLR_(G). For studies using TIRC7 protein, serialdilutions (1:200, 1:300, 1:600) of the dialyzed in vitro translatedTIRC7 protein were added to MLRs and inhibition was measured by³H-thymidine incorporation. The cytokine expression for IL-2, IL-4 andinterferon-g were detected in culture supernatants by ELISA. Commercialkits were used for IL-2 (Laboserv), IL-4 (Laboserv) and IFN-g(Medgenix). The anti-TIRC7 antibodies inhibited T cell proliferation ina dose dependent manner. Inhibition was diminished when the respectivepeptides were added to the reaction to specifically neutralize theantibody. The antibodies had no effect when added 24 h-72 h afterinitiating the MLR, indicating that the TIRC7 mediated signal wasspecific for an early event in the T cell response. These threeanti-TIRC7 antibodies also caused efficient inhibition of T-cellactivation induced by ConA, PHA, and OKT3-mAb, respectively.

[0138] Membrane proteins associated with T cell activation are ofteninvolved in ligand-receptor interactions that can be blocked byexogenous soluble protein, as has been demonstrated by blocking ofCD28/B7 interaction with the soluble protein CTLA4Ig (Linsley et al.,1992). In vitro translated TIRC7 protein was therefore tested for itsability to inhibit the MLR by adding it to MLR cultures at time 0 atdilutions of 1:200, 1:300, 1:600. 2 mg of TIRC7 cDNA were translated inan in vitro translation TNT lysate system (Promega) containing ³⁵Smethionine (ICN). The product was visualized by SDS-page (11%) andautoradiography. For MLR inhibition experiments, TIRC7 protein wassynthesized in vitro in the presence of microsomal membranes. The invitro translation mixture was then suspended in 500 μl PBS and dialyzedagainst PBS for 24 h. As shown in FIG. 5B, exogenous TIRC7 proteinsignificantly suppressed the proliferation of alloreactive T cells in adose-dependent manner. In control experiments, no inhibition was seenusing either vector alone (FIG. 5B) or another unrelated cDNA in the invitro translation reaction mixture.

Example 3

[0139] Targeting of the TIRC7 Mediated Signal Inhibits Th1 SpecificCytokine Expression, which is Reversed by Exogenous rIL-2

[0140] To further differentiate whether signals mediated by TIRC7differentially affect T cell subsets, human T cells were challenged witheither OKT3-mAb, ConA or PHA, and the cytokine profiles specific for Th1and Th2 lymphocyte subsets were analyzed in the presence and absence ofanti-TIRC7 antibodies. As shown in FIG. 5C, a significant decrease ofthe Th1-specific cytokines IL-2 and IFN-g was observed at 48 h in allcultures of PHA stimulated lymphocytes. Of the three mitogens tested,the IL-2 and IFN-g downregulation occurred in ConA and PHA activatedcultures at 24 h, whereas the OKT3-mAb stimulated T cells showed asignificant decrease of IL-2 at 24 h but of IFN-g only after 48 h. Nodownregulation of TL-4 production, which is specific for Th2lymphocytes, was observed in any of the mitogen activated T cells after24 and 48 h. Remarkably, exogenous addition of recombinant IL-2 tomitogen-activated cultures incubated with anti-TIRC7 antibodiesreconstituted the diminished T cell proliferation (FIG. 5D).

Example 4

[0141] TIRC7 Antibody Targeting Significantly Prolongs Renal AllograftSurvival in vivo

[0142] The effect of modulating the TIRC7 mediated signal was studied inan animal model featuring kidney transplantation from Wistar Furth toLewis rats. Male inbred rats 200-250 g (Harlan Winkelmann, Germany) wereused throughout the experiment. Wistar Furth rats (WF, RT1^(u)) weregrafted into bilaterally nephrectomized Lewis rats (LEW, RT1¹) usingmicrosurgical techniques; ischemic time was 30±5 min. Cryostat sectionswere fixed in formalin. The fixed tissue was paraffin embedded, andtissue sections were stained with hematoxylin and eosin. In initialexperiments, anti-human TIRC7 antibodies were tested for their abilityto inhibit the proliferation of

[0143] Lewis rat lymphocytes stimulated with irradiated Wistar Furth ratlymphocytes in vitro. Ab73 was shown to profoundly block rat T cellproliferation. In kidney transplant experiments, animals remained eitheruntreated (n=7), received preimmune rabbit serum (n=7) or were treatedwith anti-TIRC7 antibody Ab73 (n=7), 2 h before, directly after and ondays 1, 2, 4 and 6 after transplantation. No side effects except fortransient mild diarrhea were observed in the anti-TIRC7 antibody treatedgroup. Anti-TIRC7 antibody significantly prolonged the graft survivaltime of treated animals (p<0, 001). Six of seven allografts of theanti-TIRC7 treated animals remained functional for more than 45 daysafter completion of antibody administration. One animal treated withanti-TIRC7 antibody died at day 21. In contrast, all animals in bothcontrol groups died of renal failure by day 7 to 9 after transplantation(FIG. 6). Histological examination of kidney grafts from two additionalantibody treated animals sacrificed at day 7 post-transplantationdemonstrated very mild lymphocytic infiltration but no signs of tissuenecrosis (FIG. 7B). In contrast, kidney grafts from control animalsdisplayed remarkable evidence of acute graft rejection including diffusemononuclear cell infiltrates as well as extensive areas of necrosis(FIG. 7A).

[0144] Summary: TIRC7 Is a Novel T-cell Accessory Protein

[0145] TIRC7 represents a novel protein that plays an essential role inT cell activation. Early after stimulation of the T cell receptor thelevel of TIRC7 mRNA is transiently increased. This increase inexpression appears to rely on an IL-2 dependent pathway, as upregulationof TIRC7 mRNA is also observed after incubation of T cells with IL-2 andblockade of TIRC7 upregulation is achieved with cyclosporine A. T cellstimulation by mitogens such as ConA or PHA consistently fail toupregulate TIRC7 expression. The pattern of tissue expression suggeststhat TIRC7 is a product of mature lymphoid cells, as TIRC7 is expressedin all lymphoid tissues with low expression only in thymus, bone marrowand fetal liver. The TIRC7 protein is predominantly expressed on thecell membrane, consistent with a target for an external ligand. Theseven transmembrane domain structure predicts three extracellular loopsand an extracellularly oriented carboxy terminus.

[0146] Anti-TIRC7 antibodies directed against the extracellular domains,but not those recognizing predicted intracellular domains of theprotein, are able to efficiently suppress the proliferation of T cellsin response to alloactivation in a mixed lymphocyte culture or inresponse to mitogens. The inhibitory effect of anti-TIRC7 antibodies onT cells induced by a variety of different stimulatory pathways suggeststhat TIRC7 plays a central role in T cell activation. Moreover,inhibition of T cell proliferation in MLR by antibody targeting of TIRC7suggest the existence of a ligand specifically interacting with TIRC7.Support for this hypothesis is provided by the dose-dependent inhibitionof T cell proliferation in a MLR in the presence of soluble in vitrotranslated TIRC7 protein.

[0147] TIRC7 shares 38% amino acid homology with J6B7, a proteinisolated from a mouse T cell line (Lee et al., 1990). Like TIRC7, J6B7exhibits considerable homology to the putative rat H⁺-ATPase subunitVPP116 (Manolson et al., 1992). In vitro translated J6B7 protein wasdemonstrated to inhibit mouse T cell proliferation in a MLR by 89%,which is comparable with the results obtained with in vitro translatedsoluble TIRC7 protein in human MLR in the present study.

[0148] Antibody targeting of TIRC7 has a selective inhibitory effect onthe Th1 lymphocyte subset, as evidenced by the inhibition of IL-2 andIFN-g, but not IL-4, cytokine production. With anti-TIRC7 antibodytreatment the cells appear to remain in an unresponsive, but functional,state since exogenous recombinant IL-2 reversed the antiproliferativeeffect of the anti-TIRC7 antibodies. The ability of an anti-TIRC7antibody to prevent allograft rejection in the in vivo model of ratkidney transplantation reflects the findings obtained in the in vitrostudies. Moreover, the effects of antibody targeting of TIRC7 are quitesimilar to those observed by targeting of costimulatory molecules. TIRC7does not share structural or sequence homology with any of the known Tcell accessory molecules. Thus, TIRC7 may participitate in a distinctsignaling pathway induced early in the course of T cell activation.

[0149] Given the functional similarities between TIRC7 and the known Tcell accessory molecules, it is expected that the structural novelty ofTIRC7 will contribute to the understanding of distinct mechanisms in theT cell response. Moreover, the striking capacity of anti-TIRC7 antibodyto significantly prolong allograft survival in vivo provide a novelapproach for a selective inhibition of undesired T cell activation inhuman organ transplantation and autoimmune diseases.

[0150] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

[0151] References

[0152] Abbas, Nature 383 (1996); 787-793

[0153] Bachmann, Immunity 7 (1997), 549-557

[0154] Blazar J. Immunol. 157 (1996), 3250-3259

[0155] Bluestone, Immunity 2 (1995), 555-559

[0156] Bowman, J. Biol. Chem. 263 (1988), 13994-14001

[0157] Cosimi, Transplantation 32 (1981), 535-539

[0158] Crabtree, Science 243 (1989), 355-361

[0159] Kirk, Proc. Natl. Acad. Sci. USA 94 (1997), 8789-8794

[0160] Kojima, J. Biol. Chem. 271 (1996), 12327-12332

[0161] Larsen, Nature 381 (1996), 434-438

[0162] Lazarovits, Nature 380 (1996), 717-720

[0163] Lee, Mol. Immunol. 27 (1990), 1137-1144

[0164] Lenschow, Annu. Rev. Immunol. 14 (1996), 233-58

[0165] Lenschow, Science 257 (1992), 789-792

[0166] Linsley, Annu. Rev. Immunol. 11 (1993), 191-212

[0167] Linsley, Science 257 (1992), 792-95

[0168] Li, Biochem. Biophy. Res. Commun. 218 (1996), 813-821

[0169] Manolson, J. Exp. Biol. 172 (1992), 105-112

[0170] Manolson, . Biol. Chem. 269 (1994) J, 14064-14074

[0171] Mohammed, J. Exp. Med. 181 (1995), 1869-1874

[0172] Peng, J. Biol. Chem. 269 (1994), 17262-17266

[0173] Perez, Immunity 6 (1997), 411-417

[0174] Perin, J. Biol. Chem. 266 (1991), 3877-3881

[0175] Schreiber, Immunology Today 13 (1992), 136-42

[0176] Schülein, J. Biol. Chem. 271 (1996), 28844-28852

[0177] Schwartz, Cell 71 (1992), 1065-1068

[0178] Solioz, J. Biol. Chem. 269 (1994), 9453-9459

[0179] Turka, Proc. Natl. Acad. Sci. USA 89 (1992), 11102-11105

[0180] Walunas, Immunity 1 (1994), 405-413

[0181] Xu, Immunity 1 (1994), 423-431

1 14 1 2488 DNA Homo sapiens CDS (537)..(2378) 1 ggacgcccct gctccaggcccccggggggc cgcaccagga cctgagggtc aagtgagtga 60 gggatgacct catgccctttctggccagcc cagaacccct ggccagtcgc tgggctgggc 120 caggctgagc tccgactccttgtccagtgc tctccccagg ctggccccgc ctcctccttc 180 aggcccggaa cttcccacagtcccaagccc tagccctagg gggttctcct cttctggtcc 240 tgcccgggag gcctcctgccttcccctgtg ggcagggcca gtgtgcccaa ttgcccgatt 300 gcccgtgctg ggcagggtcctgcccggggg gcctggtggg ggaggcaggg caggaggttg 360 gagcagccct gcccagccccgtggccgcca gctttgtggc aggtgccgtg gagccccaca 420 aggcccctgc cctagagcgcctgctctgga gggcctgccg cggcttcctc attgccagct 480 tcagggagct ggagcagccgctggagcacc ccgtgacggg cgagccagcc acgtgg 536 atg acc ttc ctc atc tcc tactgg ggt gag cag atc gga cag aag atc 584 Met Thr Phe Leu Ile Ser Tyr trpGly Glu Gln Ile Gly Gln Lys Ile 1 5 10 15 cgc aag atc acg gac tgc ttccac tgc cac gtc ttc ccg ttt ctg cag 632 Arg Lys Ile Thr Asp Cys Phe HisCys His Val Phe Pro Phe Leu Gln 20 25 30 cag gag gag gcc cgc ctc ggg gccctg cag cag ctg caa cag cag agc 680 Gln Glu Glu Ala Arg Leu Gly Ala LeuGln Gln Leu Gln Gln Gln Ser 35 40 45 cag gag ctg cag gag gtc ctc ggg gagaca gag cgg ttc ctg agc cag 728 Gln Glu Leu Gln Glu Val Leu Gly Glu ThrGlu Arg Phe Leu Ser Gln 50 55 60 gtg cta ggc cgg gtg ctg cag ctg ctg ccgcca ggg cag gtg cag gtc 776 Val Leu Gly Arg Val Leu Gln Leu Leu Pro ProGly Gln Val Gln Val 65 70 75 80 cac aag atg aag gcc gtg tac ctg gcc ctgaac cag tgc agc gtg agc 824 His Lys Met Lys Ala Val Tyr Leu Ala Leu AsnGln Cys Ser Val Ser 85 90 95 acc acg cac aag tgc ctc att gcc gag gcc tggtgc tct gtg cga gac 872 Thr Thr His Lys Cys Leu Ile Ala Glu Ala trp CysSer Val Arg Asp 100 105 110 ctg ccc gcc ctg cag gag gcc ctg cgg gac agctcg atg gag gag gga 920 Leu Pro Ala Leu Gln Glu Ala Leu Arg Asp Ser SerMet Glu Glu Gly 115 120 125 gtg agt gcc gtg gct cac cgc atc ccc tgc cgggac atg ccc ccc aca 968 Val Ser Ala Val Ala His Arg Ile Pro Cys Arg AspMet Pro Pro Thr 130 135 140 ctc atc cgc acc aac cgc ttc acg gcc agc ttccag ggc atc gtg gat 1016 Leu Ile Arg Thr Asn Arg Phe Thr Ala Ser Phe GlnGly Ile Val Asp 145 150 155 160 gcc tac ggc gtg ggc cgc tac cag gag gtcaac ccc gct ccc tac acc 1064 Ala Tyr Gly Val Gly Arg Tyr Gln Glu Val AsnPro Ala Pro Tyr Thr 165 170 175 atc atc acc ttc ccc ttc ctg ttt gct gtgatg ttc ggg gat gtg ggc 1112 Ile Ile Thr Phe Pro Phe Leu Phe Ala Val MetPhe Gly Asp Val Gly 180 185 190 cac ggg ctg ctc atg ttc ctc ttc gcc ctggcc atg gtc ctt gcg gag 1160 His Gly Leu Leu Met Phe Leu Phe Ala Leu AlaMet Val Leu Ala Glu 195 200 205 aac cga ccg gct gtg aag gcc gcg cag aacgag atc tgg cag act ttc 1208 Asn Arg Pro Ala Val Lys Ala Ala Gln Asn GluIle trp Gln Thr Phe 210 215 220 ttc agg ggc cgc tac ctg ctc ctg ctt atgggc ctg ttc tcc atc tac 1256 Phe Arg Gly Arg Tyr Leu Leu Leu Leu Met GlyLeu Phe Ser Ile Tyr 225 230 235 240 acc ggc ttc atc tac aac gag tgc ttcagt cgc gcc acc agc atc ttc 1304 Thr Gly Phe Ile Tyr Asn Glu Cys Phe SerArg Ala Thr Ser Ile Phe 245 250 255 ccc tcg ggc tgg agt gtg gcc gcc atggcc aac cag tct ggc tgg agt 1352 Pro Ser Gly trp Ser Val Ala Ala Met AlaAsn Gln Ser Gly trp Ser 260 265 270 gat gca ttc ctg gcc cag cac acg atgctt acc ctg gat ccc aac gtc 1400 Asp Ala Phe Leu Ala Gln His Thr Met LeuThr Leu Asp Pro Asn Val 275 280 285 acc ggt gtc ttc ctg gga ccc tac cccttt ggc atc gat cct att tgg 1448 Thr Gly Val Phe Leu Gly Pro Tyr Pro PheGly Ile Asp Pro Ile trp 290 295 300 agc ctg gct gcc aac cac ttg agc ttcctc aac tcc ttc aag atg aag 1496 Ser Leu Ala Ala Asn His Leu Ser Phe LeuAsn Ser Phe Lys Met Lys 305 310 315 320 atg tcc gtc atc ctg ggc gtc gtgcac atg gcc ttt ggg gtg gtc ctc 1544 Met Ser Val Ile Leu Gly Val Val HisMet Ala Phe Gly Val Val Leu 325 330 335 gga gtc ttc aac cac gtg cac tttggc cag agg cac cgg ctg ctg ctg 1592 Gly Val Phe Asn His Val His Phe GlyGln Arg His Arg Leu Leu Leu 340 345 350 gag acg ctg ccg gag ctc acc ttcctg ctg gga ctc ttc ggt tac ctc 1640 Glu Thr Leu Pro Glu Leu Thr Phe LeuLeu Gly Leu Phe Gly Tyr Leu 355 360 365 gtg ttc cta gtc atc tac aag tggctg tgt gtc tgg gct gcc agg gcc 1688 Val Phe Leu Val Ile Tyr Lys trp LeuCys Val trp Ala Ala Arg Ala 370 375 380 gcc tcg gcc ccc agc atc ctc atccac ttc atc aac atg ttc ctc ttc 1736 Ala Ser Ala Pro Ser Ile Leu Ile HisPhe Ile Asn Met Phe Leu Phe 385 390 395 400 tcc cac agc ccc agc aac aggctg ctc tac ccc cgg cag gag gtg gtc 1784 Ser His Ser Pro Ser Asn Arg LeuLeu Tyr Pro Arg Gln Glu Val Val 405 410 415 cag gcc acg ctg gtg gtc ctggcc ttg gcc atg gtg ccc atc ctg ctg 1832 Gln Ala Thr Leu Val Val Leu AlaLeu Ala Met Val Pro Ile Leu Leu 420 425 430 ctt ggc aca ccc ctg cac ctgctg cac cgc cac cgc cgc cgc ctg cgg 1880 Leu Gly Thr Pro Leu His Leu LeuHis Arg His Arg Arg Arg Leu Arg 435 440 445 agg agg ccc gct gac cga caggag gaa aac aag gcc ggg ttg ctg gac 1928 Arg Arg Pro Ala Asp Arg Gln GluGlu Asn Lys Ala Gly Leu Leu Asp 450 455 460 ctg cct gac gca tct gtg aatggc tgg agc tcc gat gag gaa aag gca 1976 Leu Pro Asp Ala Ser Val Asn Glytrp Ser Ser Asp Glu Glu Lys Ala 465 470 475 480 ggg ggc ctg gat gat gaagag gag gcc gag ctc gtc ccc tcc gag gtg 2024 Gly Gly Leu Asp Asp Glu GluGlu Ala Glu Leu Val Pro Ser Glu Val 485 490 495 ctc atg cac cag gcc atccac acc atc gag ttc tgc ctg ggc tgc gtc 2072 Leu Met His Gln Ala Ile HisThr Ile Glu Phe Cys Leu Gly Cys Val 500 505 510 tcc aac acc gcc tcc tacctg cgc ctg tgg gcc ctg agc ctg gcc cac 2120 Ser Asn Thr Ala Ser Tyr LeuArg Leu trp Ala Leu Ser Leu Ala His 515 520 525 gcc cag ctg tcc gag gttctg tgg gcc atg gtg atg cgc ata ggc ctg 2168 Ala Gln Leu Ser Glu Val Leutrp Ala Met Val Met Arg Ile Gly Leu 530 535 540 ggc ctg ggc cgg gag gtgggc gtg gcg gct gtg gtg ctg gtc ccc atc 2216 Gly Leu Gly Arg Glu Val GlyVal Ala Ala Val Val Leu Val Pro Ile 545 550 555 560 ttt gcc gcc ttt gccgtg atg acc gtg gct atc ctg ctg gtg atg gag 2264 Phe Ala Ala Phe Ala ValMet Thr Val Ala Ile Leu Leu Val Met Glu 565 570 575 gga ctc tca gcc ttcctg cac gcc ctg cgg ctg cac tgg gtg gaa ttc 2312 Gly Leu Ser Ala Phe LeuHis Ala Leu Arg Leu His trp Val Glu Phe 580 585 590 cag aac aag ttc tactca ggc acg ggc tac aag ctg agt ccc ttc acc 2360 Gln Asn Lys Phe Tyr SerGly Thr Gly Tyr Lys Leu Ser Pro Phe Thr 595 600 605 ttc gct gcc aca gatgac tagggcccac tgcaggtcct gccagacctc 2408 Phe Ala Ala Thr Asp Asp 610cttcctgacc tctgaggcag gagaggaata aagacggtcc gccctggcaa aaaaaaaaaa 2468aaaaaaaaaa aaaaaaaaaa 2488 2 614 PRT Homo sapiens 2 Met Thr Phe Leu IleSer Tyr trp Gly Glu Gln Ile Gly Gln Lys Ile 1 5 10 15 Arg Lys Ile ThrAsp Cys Phe His Cys His Val Phe Pro Phe Leu Gln 20 25 30 Gln Glu Glu AlaArg Leu Gly Ala Leu Gln Gln Leu Gln Gln Gln Ser 35 40 45 Gln Glu Leu GlnGlu Val Leu Gly Glu Thr Glu Arg Phe Leu Ser Gln 50 55 60 Val Leu Gly ArgVal Leu Gln Leu Leu Pro Pro Gly Gln Val Gln Val 65 70 75 80 His Lys MetLys Ala Val Tyr Leu Ala Leu Asn Gln Cys Ser Val Ser 85 90 95 Thr Thr HisLys Cys Leu Ile Ala Glu Ala trp Cys Ser Val Arg Asp 100 105 110 Leu ProAla Leu Gln Glu Ala Leu Arg Asp Ser Ser Met Glu Glu Gly 115 120 125 ValSer Ala Val Ala His Arg Ile Pro Cys Arg Asp Met Pro Pro Thr 130 135 140Leu Ile Arg Thr Asn Arg Phe Thr Ala Ser Phe Gln Gly Ile Val Asp 145 150155 160 Ala Tyr Gly Val Gly Arg Tyr Gln Glu Val Asn Pro Ala Pro Tyr Thr165 170 175 Ile Ile Thr Phe Pro Phe Leu Phe Ala Val Met Phe Gly Asp ValGly 180 185 190 His Gly Leu Leu Met Phe Leu Phe Ala Leu Ala Met Val LeuAla Glu 195 200 205 Asn Arg Pro Ala Val Lys Ala Ala Gln Asn Glu Ile trpGln Thr Phe 210 215 220 Phe Arg Gly Arg Tyr Leu Leu Leu Leu Met Gly LeuPhe Ser Ile Tyr 225 230 235 240 Thr Gly Phe Ile Tyr Asn Glu Cys Phe SerArg Ala Thr Ser Ile Phe 245 250 255 Pro Ser Gly trp Ser Val Ala Ala MetAla Asn Gln Ser Gly trp Ser 260 265 270 Asp Ala Phe Leu Ala Gln His ThrMet Leu Thr Leu Asp Pro Asn Val 275 280 285 Thr Gly Val Phe Leu Gly ProTyr Pro Phe Gly Ile Asp Pro Ile trp 290 295 300 Ser Leu Ala Ala Asn HisLeu Ser Phe Leu Asn Ser Phe Lys Met Lys 305 310 315 320 Met Ser Val IleLeu Gly Val Val His Met Ala Phe Gly Val Val Leu 325 330 335 Gly Val PheAsn His Val His Phe Gly Gln Arg His Arg Leu Leu Leu 340 345 350 Glu ThrLeu Pro Glu Leu Thr Phe Leu Leu Gly Leu Phe Gly Tyr Leu 355 360 365 ValPhe Leu Val Ile Tyr Lys trp Leu Cys Val trp Ala Ala Arg Ala 370 375 380Ala Ser Ala Pro Ser Ile Leu Ile His Phe Ile Asn Met Phe Leu Phe 385 390395 400 Ser His Ser Pro Ser Asn Arg Leu Leu Tyr Pro Arg Gln Glu Val Val405 410 415 Gln Ala Thr Leu Val Val Leu Ala Leu Ala Met Val Pro Ile LeuLeu 420 425 430 Leu Gly Thr Pro Leu His Leu Leu His Arg His Arg Arg ArgLeu Arg 435 440 445 Arg Arg Pro Ala Asp Arg Gln Glu Glu Asn Lys Ala GlyLeu Leu Asp 450 455 460 Leu Pro Asp Ala Ser Val Asn Gly trp Ser Ser AspGlu Glu Lys Ala 465 470 475 480 Gly Gly Leu Asp Asp Glu Glu Glu Ala GluLeu Val Pro Ser Glu Val 485 490 495 Leu Met His Gln Ala Ile His Thr IleGlu Phe Cys Leu Gly Cys Val 500 505 510 Ser Asn Thr Ala Ser Tyr Leu ArgLeu trp Ala Leu Ser Leu Ala His 515 520 525 Ala Gln Leu Ser Glu Val Leutrp Ala Met Val Met Arg Ile Gly Leu 530 535 540 Gly Leu Gly Arg Glu ValGly Val Ala Ala Val Val Leu Val Pro Ile 545 550 555 560 Phe Ala Ala PheAla Val Met Thr Val Ala Ile Leu Leu Val Met Glu 565 570 575 Gly Leu SerAla Phe Leu His Ala Leu Arg Leu His trp Val Glu Phe 580 585 590 Gln AsnLys Phe Tyr Ser Gly Thr Gly Tyr Lys Leu Ser Pro Phe Thr 595 600 605 PheAla Ala Thr Asp Asp 610 3 13 PRT Artificial Sequence synthetic peptidederived from Homo sapiens 3 Arg Arg Pro Ala Asp Arg Gln Glu Glu Asn LysAla Gly 1 5 10 4 14 PRT Artificial Sequence synthetic peptide derivedfrom Homo sapiens 4 Ser Ser Asp Glu Glu Lys Ala Gly Gly Leu Asp Asp GluGlu 1 5 10 5 14 PRT Artificial Sequence synthetic peptide derived fromHomo sapiens 5 Val Glu Phe Gln Asn Lys Phe Tyr Ser Gly Thr Gly Tyr Lys 15 10 6 16 PRT Artificial Sequence synthetic peptide derived from Homosapiens 6 Ser Gly Thr Gly Tyr Lys Leu Ser Pro Phe Thr Phe Ala Ala ThrAsp 1 5 10 15 7 14 PRT Artificial Sequence synthetic peptide derivedfrom Homo sapiens 7 Asp Leu Pro Asp Ala Ser Val Asn Gly trp Ser Ser AspGlu 1 5 10 8 15 PRT Artificial Sequence synthetic peptide derived fromHomo sapiens 8 Gln Gln Ala Leu Arg Asp Ser Ser Met Glu Glu Gly Val SerAla 1 5 10 15 9 14 PRT Artificial Sequence synthetic peptide derivedfrom Homo sapiens 9 Ala Asn Gln Ser Gly trp Ser Asp Ala Phe Leu Ala GlnHis 1 5 10 10 14 DNA Artificial Sequence synthetic oligonucleotide - PCRPrimer 10 gacggaacag cttc 14 11 13 DNA Artificial Sequence syntheticoligonucleotide - PCR Primer 11 tgcgtctggt tct 13 12 2488 DNA Homosapiens CDS (537)..(2378) 12 ggacgcccct gctccaggcc cccggggggc cgcaccaggacctgagggtc aagtgagtga 60 gggatgacct catgcccttt ctggccagcc cagaacccctggccagtcgc tgggctgggc 120 caggctgagc tccgactcct tgtccagtgc tctccccaggctggccccgc ctcctccttc 180 aggcccggaa cttcccacag tcccaagccc tagccctagggggttctcct cttctggtcc 240 tgcccgggag gcctcctgcc ttcccctgtg ggcagggccagtgtgcccaa ttgcccgatt 300 gcccgtgctg ggcagggtcc tgcccggggg gcctggtgggggaggcaggg caggaggttg 360 gagcagccct gcccagcccc gtggccgcca gctttgtggcaggtgccgtg gagccccaca 420 aggcccctgc cctagagcgc ctgctctgga gggcctgccgcggcttcctc attgccagct 480 tcagggagct ggagcagccg ctggagcacc ccgtgacgggcgagccagcc acgtgg 536 atg acc ttc ctc atc tcc tac tgg ggt gag cag atcgga cag aag atc 584 Met Thr Phe Leu Ile Ser Tyr trp Gly Glu Gln Ile GlyGln Lys Ile 1 5 10 15 cgc aag atc acg gac tgc ttc cac tgc cac gtc ttcccg ttt ctg cag 632 Arg Lys Ile Thr Asp Cys Phe His Cys His Val Phe ProPhe Leu Gln 20 25 30 cag gag gag gcc cgc ctc ggg gcc ctg cag cag ctg caacag cag agc 680 Gln Glu Glu Ala Arg Leu Gly Ala Leu Gln Gln Leu Gln GlnGln Ser 35 40 45 cag gag ctg cag gag gtc ctc ggg gag aca gag cgg ttc ctgagc cag 728 Gln Glu Leu Gln Glu Val Leu Gly Glu Thr Glu Arg Phe Leu SerGln 50 55 60 gtg cta ggc cgg gtg ctg cag ctg ctg ccg cca ggg cag gtg caggtc 776 Val Leu Gly Arg Val Leu Gln Leu Leu Pro Pro Gly Gln Val Gln Val65 70 75 80 cac aag atg aag gcc gtg tac ctg gcc ctg aac cag tgc agc gtgagc 824 His Lys Met Lys Ala Val Tyr Leu Ala Leu Asn Gln Cys Ser Val Ser85 90 95 acc acg cac aag tgc ctc att gcc gag gcc tgg tgc tct gtg cga gac872 Thr Thr His Lys Cys Leu Ile Ala Glu Ala trp Cys Ser Val Arg Asp 100105 110 ctg ccc gcc ctg cag gag gcc ctg cag gac agc tcg atg gag gag gga920 Leu Pro Ala Leu Gln Glu Ala Leu Gln Asp Ser Ser Met Glu Glu Gly 115120 125 gtg agt gcc gtg gct cac cgc atc ccc tgc cgg gac atg ccc ccc aca968 Val Ser Ala Val Ala His Arg Ile Pro Cys Arg Asp Met Pro Pro Thr 130135 140 ctc atc cgc acc aac cgc ttc acg gcc agc ttc cag ggc atc gtg gat1016 Leu Ile Arg Thr Asn Arg Phe Thr Ala Ser Phe Gln Gly Ile Val Asp 145150 155 160 gcc tac ggc gtg ggc cgc tac cag gag gtc aac ccc gct ccc tacacc 1064 Ala Tyr Gly Val Gly Arg Tyr Gln Glu Val Asn Pro Ala Pro Tyr Thr165 170 175 atc atc acc ttc ccc ttc ctg ttt gct gtg atg ttc ggg gat gtgggc 1112 Ile Ile Thr Phe Pro Phe Leu Phe Ala Val Met Phe Gly Asp Val Gly180 185 190 cac ggg ctg ctc atg ttc ctc ttc gcc ctg gcc atg gtc ctt gcggag 1160 His Gly Leu Leu Met Phe Leu Phe Ala Leu Ala Met Val Leu Ala Glu195 200 205 aac cga ccg gct gtg aag gcc gcg cag aac gag atc tgg cag actttc 1208 Asn Arg Pro Ala Val Lys Ala Ala Gln Asn Glu Ile trp Gln Thr Phe210 215 220 ttc agg ggc cgc tac ctg ctc ctg ctt atg ggc ctg ttc tcc atctac 1256 Phe Arg Gly Arg Tyr Leu Leu Leu Leu Met Gly Leu Phe Ser Ile Tyr225 230 235 240 acc ggc ttc atc tac aac gag tgc ttc agt cgc gcc acc agcatc ttc 1304 Thr Gly Phe Ile Tyr Asn Glu Cys Phe Ser Arg Ala Thr Ser IlePhe 245 250 255 ccc tcg ggc tgg agt gtg gcc gcc atg gcc aac cag tct ggctgg agt 1352 Pro Ser Gly trp Ser Val Ala Ala Met Ala Asn Gln Ser Gly trpSer 260 265 270 gat gca ttc ctg gcc cag cac acg atg ctt acc ctg gat cccaac gtc 1400 Asp Ala Phe Leu Ala Gln His Thr Met Leu Thr Leu Asp Pro AsnVal 275 280 285 acc ggt gtc ttc ctg gga ccc tac ccc ttt ggc atc gat cctatt tgg 1448 Thr Gly Val Phe Leu Gly Pro Tyr Pro Phe Gly Ile Asp Pro Iletrp 290 295 300 agc ctg gct gcc aac cac ttg agc ttc ctc aac tcc ttc aagatg aag 1496 Ser Leu Ala Ala Asn His Leu Ser Phe Leu Asn Ser Phe Lys MetLys 305 310 315 320 atg tcc gtc atc ctg ggc gtc gtg cac atg gcc ttt ggggtg gtc ctc 1544 Met Ser Val Ile Leu Gly Val Val His Met Ala Phe Gly ValVal Leu 325 330 335 gga gtc ttc aac cac gtg cac ttt ggc cag agg cac cggctg ctg ctg 1592 Gly Val Phe Asn His Val His Phe Gly Gln Arg His Arg LeuLeu Leu 340 345 350 gag acg ctg ccg gag ctc acc ttc ctg ctg gga ctc ttcggt tac ctc 1640 Glu Thr Leu Pro Glu Leu Thr Phe Leu Leu Gly Leu Phe GlyTyr Leu 355 360 365 gtg ttc cta gtc atc tac aag tgg ctg tgt gtc tgg gctgcc agg gcc 1688 Val Phe Leu Val Ile Tyr Lys trp Leu Cys Val trp Ala AlaArg Ala 370 375 380 gcc tcg gcc ccc agc atc ctc atc cac ttc atc aac atgttc ctc ttc 1736 Ala Ser Ala Pro Ser Ile Leu Ile His Phe Ile Asn Met PheLeu Phe 385 390 395 400 tcc cac agc ccc agc aac agg ctg ctc tac ccc cggcag gag gtg gtc 1784 Ser His Ser Pro Ser Asn Arg Leu Leu Tyr Pro Arg GlnGlu Val Val 405 410 415 cag gcc acg ctg gtg gtc ctg gcc ttg gcc atg gtgccc atc ctg ctg 1832 Gln Ala Thr Leu Val Val Leu Ala Leu Ala Met Val ProIle Leu Leu 420 425 430 ctt ggc aca ccc ctg cac ctg ctg cac cgc cac cgccgc cgc ctg cgg 1880 Leu Gly Thr Pro Leu His Leu Leu His Arg His Arg ArgArg Leu Arg 435 440 445 agg agg ccc gct gac cga cag gag gaa aac aag gccggg ttg ctg gac 1928 Arg Arg Pro Ala Asp Arg Gln Glu Glu Asn Lys Ala GlyLeu Leu Asp 450 455 460 ctg cct gac gca tct gtg aat ggc tgg agc tcc gatgag gaa aag gca 1976 Leu Pro Asp Ala Ser Val Asn Gly trp Ser Ser Asp GluGlu Lys Ala 465 470 475 480 ggg ggc ctg gat gat gaa gag gag gcc gag ctcgtc ccc tcc gag gtg 2024 Gly Gly Leu Asp Asp Glu Glu Glu Ala Glu Leu ValPro Ser Glu Val 485 490 495 ctc atg cac cag gcc atc cac acc atc gag ttctgc ctg ggc tgc gtc 2072 Leu Met His Gln Ala Ile His Thr Ile Glu Phe CysLeu Gly Cys Val 500 505 510 tcc aac acc gcc tcc tac ctg cgc ctg tgg gccctg agc ctg gcc cac 2120 Ser Asn Thr Ala Ser Tyr Leu Arg Leu trp Ala LeuSer Leu Ala His 515 520 525 gcc cag ctg tcc gag gtt ctg tgg gcc atg gtgatg cgc ata ggc ctg 2168 Ala Gln Leu Ser Glu Val Leu trp Ala Met Val MetArg Ile Gly Leu 530 535 540 ggc ctg ggc cgg gag gtg ggc gtg gcg gct gtggtg ctg gtc ccc atc 2216 Gly Leu Gly Arg Glu Val Gly Val Ala Ala Val ValLeu Val Pro Ile 545 550 555 560 ttt gcc gcc ttt gcc gtg atg acc gtg gctatc ctg ctg gtg atg gag 2264 Phe Ala Ala Phe Ala Val Met Thr Val Ala IleLeu Leu Val Met Glu 565 570 575 gga ctc tca gcc ttc ctg cac gcc ctg cggctg cac tgg gtg gaa ttc 2312 Gly Leu Ser Ala Phe Leu His Ala Leu Arg LeuHis trp Val Glu Phe 580 585 590 cag aac aag ttc tac tca ggc acg ggc tacaag ctg agt ccc ttc acc 2360 Gln Asn Lys Phe Tyr Ser Gly Thr Gly Tyr LysLeu Ser Pro Phe Thr 595 600 605 ttc gct gcc aca gat gac tagggcccactgcaggtcct gccagacctc 2408 Phe Ala Ala Thr Asp Asp 610 cttcctgacctctgaggcag gagaggaata aagacggtcc gccctggcaa aaaaaaaaaa 2468 aaaaaaaaaaaaaaaaaaaa 2488 13 614 PRT Homo sapiens 13 Met Thr Phe Leu Ile Ser Tyrtrp Gly Glu Gln Ile Gly Gln Lys Ile 1 5 10 15 Arg Lys Ile Thr Asp CysPhe His Cys His Val Phe Pro Phe Leu Gln 20 25 30 Gln Glu Glu Ala Arg LeuGly Ala Leu Gln Gln Leu Gln Gln Gln Ser 35 40 45 Gln Glu Leu Gln Glu ValLeu Gly Glu Thr Glu Arg Phe Leu Ser Gln 50 55 60 Val Leu Gly Arg Val LeuGln Leu Leu Pro Pro Gly Gln Val Gln Val 65 70 75 80 His Lys Met Lys AlaVal Tyr Leu Ala Leu Asn Gln Cys Ser Val Ser 85 90 95 Thr Thr His Lys CysLeu Ile Ala Glu Ala trp Cys Ser Val Arg Asp 100 105 110 Leu Pro Ala LeuGln Glu Ala Leu Gln Asp Ser Ser Met Glu Glu Gly 115 120 125 Val Ser AlaVal Ala His Arg Ile Pro Cys Arg Asp Met Pro Pro Thr 130 135 140 Leu IleArg Thr Asn Arg Phe Thr Ala Ser Phe Gln Gly Ile Val Asp 145 150 155 160Ala Tyr Gly Val Gly Arg Tyr Gln Glu Val Asn Pro Ala Pro Tyr Thr 165 170175 Ile Ile Thr Phe Pro Phe Leu Phe Ala Val Met Phe Gly Asp Val Gly 180185 190 His Gly Leu Leu Met Phe Leu Phe Ala Leu Ala Met Val Leu Ala Glu195 200 205 Asn Arg Pro Ala Val Lys Ala Ala Gln Asn Glu Ile trp Gln ThrPhe 210 215 220 Phe Arg Gly Arg Tyr Leu Leu Leu Leu Met Gly Leu Phe SerIle Tyr 225 230 235 240 Thr Gly Phe Ile Tyr Asn Glu Cys Phe Ser Arg AlaThr Ser Ile Phe 245 250 255 Pro Ser Gly trp Ser Val Ala Ala Met Ala AsnGln Ser Gly trp Ser 260 265 270 Asp Ala Phe Leu Ala Gln His Thr Met LeuThr Leu Asp Pro Asn Val 275 280 285 Thr Gly Val Phe Leu Gly Pro Tyr ProPhe Gly Ile Asp Pro Ile trp 290 295 300 Ser Leu Ala Ala Asn His Leu SerPhe Leu Asn Ser Phe Lys Met Lys 305 310 315 320 Met Ser Val Ile Leu GlyVal Val His Met Ala Phe Gly Val Val Leu 325 330 335 Gly Val Phe Asn HisVal His Phe Gly Gln Arg His Arg Leu Leu Leu 340 345 350 Glu Thr Leu ProGlu Leu Thr Phe Leu Leu Gly Leu Phe Gly Tyr Leu 355 360 365 Val Phe LeuVal Ile Tyr Lys trp Leu Cys Val trp Ala Ala Arg Ala 370 375 380 Ala SerAla Pro Ser Ile Leu Ile His Phe Ile Asn Met Phe Leu Phe 385 390 395 400Ser His Ser Pro Ser Asn Arg Leu Leu Tyr Pro Arg Gln Glu Val Val 405 410415 Gln Ala Thr Leu Val Val Leu Ala Leu Ala Met Val Pro Ile Leu Leu 420425 430 Leu Gly Thr Pro Leu His Leu Leu His Arg His Arg Arg Arg Leu Arg435 440 445 Arg Arg Pro Ala Asp Arg Gln Glu Glu Asn Lys Ala Gly Leu LeuAsp 450 455 460 Leu Pro Asp Ala Ser Val Asn Gly trp Ser Ser Asp Glu GluLys Ala 465 470 475 480 Gly Gly Leu Asp Asp Glu Glu Glu Ala Glu Leu ValPro Ser Glu Val 485 490 495 Leu Met His Gln Ala Ile His Thr Ile Glu PheCys Leu Gly Cys Val 500 505 510 Ser Asn Thr Ala Ser Tyr Leu Arg Leu trpAla Leu Ser Leu Ala His 515 520 525 Ala Gln Leu Ser Glu Val Leu trp AlaMet Val Met Arg Ile Gly Leu 530 535 540 Gly Leu Gly Arg Glu Val Gly ValAla Ala Val Val Leu Val Pro Ile 545 550 555 560 Phe Ala Ala Phe Ala ValMet Thr Val Ala Ile Leu Leu Val Met Glu 565 570 575 Gly Leu Ser Ala PheLeu His Ala Leu Arg Leu His trp Val Glu Phe 580 585 590 Gln Asn Lys PheTyr Ser Gly Thr Gly Tyr Lys Leu Ser Pro Phe Thr 595 600 605 Phe Ala AlaThr Asp Asp 610 14 10 PRT UNKNOWN Peptide sequence of solublefibronectin 14 Gly Pro Glu Ile Leu Asp Val Pro Ser Thr 1 5 10

What is claimed is:
 1. A polynucleotide encoding a TIRC7 membraneprotein or a biologically active fragment thereof comprising a DNAsequence selected from the group consisting of (i) DNA sequencescomprising a nucleotide sequence encoding the amino acid sequencedepicted in SEQ ID NO. 2 or SEQ ID NO. 13 from amino acid position 1 to614 or from amino acid position 1 to 601; (ii) DNA sequences comprisingthe nucleotide sequence depicted in SEQ ID NO. 1 or SEQ ID NO. 12; (ii)DNA sequences comprising a nucleotide sequence encoding a fragment orderivative of the protein encoded by the DNA sequence of (i) or (ii);and (iii) DNA sequences the complementary strand of which hybridizeswith and which is at least 70% identical to the polynucleotide asdefined in any one of (i) to (iii).
 2. A nucleic acid molecule of atleast 15 nucleotides in length hybridizing specifically with apolynucleotide of claim 1 or with a complementary strand thereof.
 3. Avector comprising the polynucleotide of claim
 1. 4. The vector of claim3, wherein said polynucleotide is operably linked to regulatorysequences allowing for the transcription and optionaly expression ofsaid nucleic acid molecule.
 5. A host cell comprising a polynucleotideof claim 1 or the vector of claim 3 or
 4. 6. A method for the productionof a TIRC7 membrane protein or a biologically active fragment thereofcomprising: (a) culturing the host of claim 5 under conditions allowingfor the expression of the protein; or (b) in vitro translation of thepolynucleotide of claim 1; and recovering the protein produced in (a) or(b).
 7. A TIRC7 membrane protein or biologically active fragment thereofencoded by the nucleic molecule of claim 1 or produced by the method ofclaim
 6. 8. An antibody specifically recognizing the protein of claim 7.9. A normal cell that has been modified to express the protein of claim7 or the antibody of claim
 8. 10. A pharmaceutical compositioncomprising a peptide or polypeptide being capable of inhibiting T-cellstimulation through the TIRC7 membrane protein and/or being recognizedby an antibody capable of inhibiting T-cell stimulation through theTIRC7 membrane protein encoded by a fragment of the polynucleotide ofclaim 1 or an antibody specifically recognizing said peptide orpolypeptide, and optionally a pharmaceutically acceptable carrier. 11.The pharmaceutical composition of claim 10, wherein said peptide orpolypeptide is in a soluble form.
 12. The pharmaceutical composition ofclaim 10 or 11, wherein said peptide or polypeptide comprises the aminoacid sequence depicted in any one of SEQ ID NOS 3 to
 9. 13. Thepharmaceutical composition of any one of claims 10 to 12 furthercomprising a second agent which inhibits T-cell stimulation.
 14. Thepharmaceutical composition of claim 13, wherein the second agent is anantibody to a receptor or ligand involved in T-cell stimulation or is asoluble form of said receptor.
 15. The pharmaceutical composition ofclaim 14, wherein said second agent blocks interaction of the CD2, CD28,CTLA4 or CD40 surface receptor with a CD2, CD28, CTLA4 or CD40 ligand.16. The pharmaceutical composition of any one of claims 10 to 15 for usein cell or organ transplantation, for the treatment of autoimmune,allergic or infectious diseases, or for the treatment of tumors.
 17. Adiagnostic composition comprising a polynucleotide of claim 1, thevector of claim 3 or 4, the cell of claim 5 or 9, the protein of claim7, or the antibody of claim 8 or a (poly)peptide or antibody as definedin any one of claims 10 to 12; and optionally suitable means fordetection.
 18. An in vitro method for inducing or maintainingunresponsiveness of a T-cell to an antigen comprising contacting theT-cell with an agent which inhibits stimulation of the T-cell through aTIRC7 membrane protein.
 19. Use of an agent which inhibits T-cellstimulation through a TIRC7 membrane protein for the preparation of apharmaceutical composition for inducing or maintaining T-cellunresponsiveness to an antigen in a subject.
 20. The method of claim 18or the use of claim 19, wherein the agent blocks an interaction of theTIRC7 membrane protein with its ligand.
 21. The method or the use ofclaim 20, wherein the agent is a peptide or polypeptide being capable ofinhibiting T-cell stimulation through the TIRC7 membrane protein and/orbeing recognized by an antibody capable of inhibiting T-cell stimulationthrough the TIRC7 membrane protein encoded by a fragment of thepolynucleotide of claim 1 or an antibody specifically recognizing saidpeptide or polypeptide.
 22. The method of any one of claims 18, 20 or 22or the use of any one of claims 19 to 21, further comprising the use ofa second agent as defined in any one of claims 13 to
 16. 23. Apharmaceutical composition comprising a first agent which stimulates aT-cell through a TIRC7 membrane protein and optionally apharmaceutically acceptable carrier.
 24. The pharmaceutical compositionof claim 23, wherein said agent is a ligand of the TIRC7 membraneprotein or is at least one anti-TIRC7 membrane protein antibody.
 25. Thepharmaceutical composition of claim 23 to 24 further comprising a secondagent which stimulates T-cell proliferation.
 26. The pharmaceuticalcomposition of claim 25, wherein said agent is IL-2, IL-4 or an agentwhich stimulates a T-cell through a CD2, CD28, CD40 or CTLA4 surfacereceptor.
 27. An in vitro method for restoring responsiveness to anantigen by a T-cell which is unresponsive to the antigen, comprisingcontacting the T-cell in the presence of the antigen with a first agentwhich stimulates the T-cell through a TIRC7 membrane protein.
 28. Themethod of claim 27, wherein the TIRC7 ligand is expressed on a cellsurface by introducing to the cell a nucleic acid molecule encoding theTIRC7 ligand in a suitable form for expression of the TIRC7 ligand onthe cell surface.
 29. The method of claim 28, wherein the cell is atumor cell.
 30. The method of any one of claims 27 to 29, furthercomprising contacting the T-cell with a second agent as defined in claim25 or
 26. 31. The method of claim 30, wherein the T-cell is contactedwith the second agent prior to being contacted with the first agent. 32.The method of claim 30 or 31, wherein a CD2, CD28, CD40 or CTLA4 ligandis expressed on the cell surface by introducing into the cell a nucleicacid molecule encoding the CD2, CD28, CD40 or CTLA4 ligand in a formsuitable for expression of said ligand on the cell surface.
 33. Use of afirst agent as defined in claim 23 to 24 for the preparation of apharmaceutical composition for stimulating a T-cell response to a tumorcell in a subject with a tumor.
 34. The use of claim 33 wherein thetumor cell is modified to express a TIRC7 ligand and/or a CD2, CD28,CD40 or CTLA4 ligand preferably on the tumor cell surface.
 35. The useof claim 33 or 34, wherein the tumor cell is obtained from the subject,modified ex vivo to form a modified tumor cell and said modified tumorcell is used for the preparation of a pharmaceutical composition whichis designed for administration to the subject.
 36. The use of any one ofclaims 33 to 35, wherein the T-cells are obtained from a subject,contacted with IL-2 or IL-4 ex vivo and said modified T-cells are usedfor the preparation of pharmaceutical composition which is designed forthe administration to the subject.
 37. The use of any one of claims 19to 22 or 33 to 35, wherein the subject is a recipient of peripheral stemcells or bone marrow transplant.
 38. The use of claim 37, wherein thepharmaceutical composition is designed for contacting with peripheralstem cells or bone marrow cell prior to transplantation into therecipient.
 39. The method of any one of claims 18, 20 to 22 or any oneof claims 27 to 32 or the use of any one of claims 19 to 22 or 33 to 36in organ graft transplantation or for the treatment of auto-immunediseases.
 40. A method for identifying T-cell activating orco-stimulating compounds or for identifying inhibitors of T-cellactivation and stimulation comprising (a) culturing T-cells in thepresence of the protein of claim 7, the antibody of claim 8, the cell ofclaim 5 or 9 or a peptide or polypeptide being capable of inhibitingT-cell stimulation through the TIRC7 membrane protein and/or beingrecognized by an antibody capable of inhibiting T-cell stimulationthrough the TIRC7 membrane protein encoded by a fragment of thepolynucleotide of claim 1 or an antibody specifically recognizing saidpeptide or polypeptide and, optionally, in the presence of a componentcapable of providing a detectable signal in response to T-cellactivation, with a compound to be screened under conditions permittinginteraction of the compound with the (poly)peptide, antibody or cell(s);and (b) detecting the presence or absence of a signal generated from theinteraction of the compound with the cells.
 41. A method for theproduction of a pharmaceutical compositions comprising the steps of themethod of claim 40 and formulating and optionally synthesizing thecompound identified in step (b) in a pharmaceutically acceptable form.42. Use of peptide or polypeptide being capable of inhibiting T-cellstimulation through the TIRC7 membrane protein and/or being recognizedby an antibody capable of inhibiting T-cell stimulation through theTIRC7 membrane protein encoded by a fragment of the polynucleotide ofclaim 1 or an antibody specifically recognizing said peptide orpolypeptide the polynucleotide of claim 1, the vector of claim 3 or 4,the protein of claim 7, the antibody of claim 8, the cell of claim 5 or9 or the compound indentified according to the method of claim 40 forthe preparation of a pharmaceutical composition for the treatment ofacute and chronic diseases, involving T-cell activation and Th1 and Th2immune response, for the treatment of acute and chronic rejection ofallo- and xeno organ transplants and bone marrow transplantation, forthe treatment of rheumatoid arthritis, lupus erythramatodes, multiplesklerosis, encephalitis, vasculitis, diabetes mellitus, pancreatitis,gastritis, thyroiditis, for the treatment of maligne disorders of T, Bor NK cells, for the treatment of asthma, lepramatosis, Helicobacterpylori associated gastritis or for the treatment of skin tumors, adrenaltumors or lung tumors.